CN101845009B - Structural carotenoid analogs for the inhibition and amelioration of disease - Google Patents

Abstract

The invention relates to structural carotenoid analogs for the inhibition and amelioration of the disease. The invention also relates to compositions comprising structural carotenoid analogs and a use thereof. The structural carotenoid analogs are used for inhibiting and/or ameliorating the occurrence of diseases associated with reactive oxygen species, reactive nitrogen species, radicals and/or non-radicals, for example, the inhibition and/or amelioration of ischemia-reperfusion injury, the inhibition and/or amelioration of liver disease, and the inhibition and/or amelioration of cancer.

Description

Be used to suppress and improve the structural carotenoid analogs of disease

The application is that application number is the dividing an application for the patented claim of " being used to suppress and improve the structural carotenoid analogs of disease " that 03823260.X, the applying date be on July 29th, 2003, denomination of invention.

Technical field

Present invention relates in general to medicine and synthetic chemistry field.More specifically, the present invention relates to the synthetic and application of the similar thing of carrotenoid.

Background technology

Cardiovascular disorder (CVD), particularly coronary artery disease (CAD) are still the dead leading reason in the U.S. and the whole world.CVD is the leading reason of whole world mortality ratio and sickness rate.Slightly reduce cardiovascular risk to moderate, thereby cause the emergency unit of acute coronary syndrome to be gone to a doctor and hospital care reduces, can produce significantly clinical and public health benefit.

Broad research to inhibitor has shown that they are elementary and the effective therapeutical agent secondary prevention cardiovascular disorder.CVD is still the dead leading reason of all races of the U.S.; About 6,000 ten thousand Americans suffer from the CVD of certain form now.If can eliminate CVD then will increase similar 7 years in the predicted life of the U.S..The since nineteen ninety-six existing institute of the absolute death toll that is caused by CVD reduces, but it is still the single maximum cause of death in the U.S., and Da Yu $3000 hundred million (comprise and having a heart attack and apoplexy) is born in its total annual health care.

Local asphyxia is that specific tissue lacks enough Oxygenated blood confessions.Local asphyxia is the basis of many acute and chronic disease situations, and said disease includes but not limited to:

● myocardial infarction, or MI

● unstable angina pectoris

● stable stenocardia

● the unexpected reclosing behind Pi Jing chamber coronary angioplasty (PTCA)

● thrombotic apoplexy (apoplexy sum 85%)

● the embolic vascular occlusion

● peripheral blood vessel is insufficient

● organ transplantation

● venous thrombosis, or DVT

● inlying catheter is inaccessible

Local asphyxia also possibly become a problem in the selectivity operation, for example: predetermined organ transplantation; Predetermined coronary artery bypass transplantation (CABG); With predetermined coronary angioplasty (PTCA) through the Pi Jing chamber.The something in common of each in these situation is the reperfusion injury phenomenon: when Oxygenated blood stream being reintroduced to previous ischemic zone, produce reactive oxygen species (ROS), unusual extra tissue injury takes place subsequently.Especially, Acute Myocardial Infarction (AMI) and acute thrombus form use in the property apoplexy thrombolytic therapy-and with PTCA carry out surgery vascular form again-usually with the perfusion again of local asphyxia cardiac muscle and/or brain.Clinical effectiveness is realized opening in early days and improving along with form the back at acute thrombus, but is not need not pay a price (i.e. " reperfusion injury ").

Present treatment allows to pour into pharmacologically active agents again, comprises recombinant tissue-type plasminogen activator (r-TPA), Eminase (APSAC), streptokinase and urokinase.Recent research has shown that early stage surgery pours into the optimal clinical result who brings after the AMI again.But U.S.'s medical centre that surgery only pours at 15-20% again can obtain, in the whole world then still less.Therefore, pharmacology pour into again being still foreseeable future suitable to clinically important.

Thromboembolism treatment is unsuccessful in the perfusion again of about 20% infraction artery.Successfully carrying out again in the dabbling artery about 15% reclosing (within 24 hours) suddenly.The measurement index of system inflammation (the for example serum level of C-reactive protein or CRP) is very relevant with these patients' clinical reclosing.As if cardiac muscle rescue effect maximum in 2-6 hour " the treatment window " after acute plaque rupture and thrombosis.Form in property or the thromboembolic stroke at acute thrombus, this treatment window even narrower is usually less than behind the thrombosis 3 hours.Administered recombinant histiotype plasminogen activator improves clinical effectiveness significantly in ischemic stroke 3 hours, but increases risk of bleeding.

During local asphyxia, many cell experience biological chemistry and pathology relevant with anoxic change, but keep potential vigor.Therefore the cell of these potential survivals is again " battlefields " in the flush phase.Local asphyxia causes that affected tissue changes, and possibly finally cause being in the contraction bands and/or the coagulation necrosis of dangerous cardiac muscle.The pathological change of local asphyxia cardiac muscle includes but not limited to:

● radical and ROS produce

● ATP forfeiture and defective ATP resynthesis

● the phosphocreatine forfeiture

● the forfeiture of extracellular potassium

● cardiac muscle produces the Disability of active tension

● cellular swelling

● oxypathy

● the forfeiture of ion stable state

● structure deteriorate

● the unstable and irregular pulse generation of electricity

● the adipose membrane superoxide

● gsh and other endogenous/exogenous antioxidant consumption (comprising vitamins C and E and carrotenoid)

Rescuing of local asphyxia cardiac muscle to irreversibly not reaching downright bad threshold as yet is the focus of intervening in the reperfusion injury.

It is a kind of iuntercellular connection of the unique types of in most of zooblast type, finding that the gap connects.They form and make the interconnected water-based passage of tenuigenin of flanking cell, and make the direct iuntercellular exchange of little (less than about 1 kilodalton) cytoplasm fraction become possibility.Two hemichannels (" connexon ") through butt joint is provided by each flanking cell are crossed over born of the same parents' external space establishment gap connection therebetween.Each hemichannel is 6 oligopolymer that connect protein molecular.

Connect protein 43 and be second kind of being found and connect the albumen group, the clone that it is coded in establishment with organize in wide expression be connected a kind of in the albumen.The gap connection that is formed by the connection protein 43 relates to growth, heart function and adjusting and controlling growth.

The common performance of CVD is an irregular pulse.It is generally acknowledged that irregular pulse is the disorder of cardiac electrical activity, it shows as heart rate or rhythm abnormality.Patients with arrhythmia possibly experience the extensive symptom from palpitaition to swoon (" fainting ").

Main connection albumen in the cardiovascular systems is for connecting protein 43.Think that in cells of vascular wall particularly connect to coordinate for the keeping of vasomotion tensile local modulation and circulation stable state be critical in the gap of cell response among the endotheliocyte.The rise of control linkage protein 43 can also help to keep the elctrical stability in the heart tissue.Keep the elctrical stability in the heart tissue can be of value to annual hundreds thousand of health of suffering from the people of some type cardiovascular disorder [for example ischemic heart disease (IHD) and irregular pulse], and can prevent that the patient who is in height irregular pulse risk from sudden cardiac death taking place.

The characteristic that it has been generally acknowledged that cancer is controlled, not unusual cell growth.Aforesaid connection protein 43 is also relevant with the cell growth control.Growth control due to the connection protein 43 maybe be relevant with gap junction communication owing to connecting protein 43.Keep, recover or increase functional clearance connecting the propagation that communication suppresses transformant.Therefore, the availability of rise and/or control linkage protein 43 can suppress and/or improve the diffusion of cancer cells potentially.

Chronic hepatic injury no matter its cause of disease how, can cause from acute and chronic inflammation to early stage fibrosis, and is finally managed scope to the venereal disease of carrying out of liver cirrhosis and hepatic diseases in late period (ESRD).The inflammatory events cascade of initial damage secondary comprises discharging cytokine and forming reactive oxygen species (ROS), activation stellate cells (HSC).HSC produces extracellular matrix components (ECM), comprises collagen, and is crucial in the process that produces hepatic fibrosis.

Hepatic diseases in late period [showing as liver cirrhosis or hepatocellular carcinoma (HCC)] is the 8th a leading reason of U.S.'s death relevant with disease.The chronic inflammatory diseases of the liver that is caused by virus infection, alcohol abuse, drug-induced toxicity, iron and copper sh and many other factorses can cause hepatic fibrosis.The by product activation Kupffer Cell of hepatocellular damage, it discharges the various kinds of cell factor, ROS (particularly including superoxide anion) and other paracrine and the autocrine factor then, and then acts on stellate cells (HSC).Think that at present the key cells in the fiber generation cascade is the cell type that HsC-is responsible for producing ECM.External evidence shows that ROS can induce the HSC cell.The level of in all chronic hepatic diseases patients, observing the indirect labels (for example thiobarbituricacid reactive specy or TBARS) of oxidative stress raises.In addition, the level of chronic hepatic diseases patient's gsh, Selenoperoxidase, superoxide-dismutase, carrotenoid and alpha-tocopherol (vitamin E) is obviously lower.The sign that provides these endogenous and/or exogenous antioxidant to reverse many chronic hepatic diseases comprises the direct measurement index that substitutes mark and hepatic fibrosis of the course of disease.Therefore, they possibly be the beneficial agentss that liver disease therapy is intervened.

Summary of the invention

In certain embodiments, the analog of using carrotenoid can suppress and/or improve the generation of treatment target disease.Can comprise with the disease of the analog of carrotenoid treatment and relate to any disease that produces reactive oxygen species and/or other radical kind (for example singlet oxygen, this is a kind of reactive oxygen species but is not radical).In certain embodiments, the water-soluble carotenoid analogue can be used to treat and relates to the disease that produces reactive oxygen species.Reactive oxygen species and other radical and non-free radical kind relate to many human diseasess to the oxidation of DNA, albumen and lipid.Radical possibly be the major cause of following illness, can make health more responsive to other diseases induced factor, can suppress endogenous defence and repair process, and/or can promote the progress of initial stage disease.Those skilled in the art use the analog of carrotenoid-comprise and consider that pharmacokinetics and pharmacodynamics-expection that medicine is sent can suppress and/or improve said illness.In first kind illness, mainly be that one organ is influenced, its evidence is in the pathology of disease, to relate to radical and/or non-free radical.These instances should not be counted as qualification, and other illness is apparent to those skilled in the art.

Head, eye, ear, N&T: the degeneration of macula relevant (ARMD), retinal detachment, hypertensive cerebral retinal diseases, uveitis, choroiditis, hyalitis, ophthalmorrhagia, degenerative retina injury, cataract generation and cataract, retinopathy of prematurity, Meniere, drug-induced ototoxicity (comprising aminoglycoside and furosemide toxicity), the infectious and special property sent out otitis, otitis media, infectivity and allergic sinusitis, Head and Neck cancer with the age;

Cns (brain and spinal cord): senile dementia (comprising Alzheimer); Niemann-Pick disease; The neurotoxin reaction; The hyperbaric oxygen effect; Parkinson's disease; Brain and trauma of spinal cord; The hypertensive cerebral cerebrovascular trauma; Apoplexy (thromboembolic states; Thrombotic and hemorrhagic); Infectious encephalitis and meningitis; Allergic encephalitis and other demyelination; Amyotrophic lateral sclerosis (ALS); Multiple sclerosis; NCL; Ataxia-telangiectasia syndrome; Aluminium; Iron and other heavy metal sh; Primary brain cancer/malignant tumour and metastatic encephaloma;

Cardiovascular: arteriosclerosis; Atherosclerosis; Peripheral vascular disease; Myocardial infarction; Chronic stable stenocardia; Unstable angina pectoris; The special property sent out surgical injury (CABG; During the PTCA); Inflammatory heart disease [weigh and influenced by it] by C-reactive protein (CRP) and myeloperoxidase (MPO); LDL oxidation (ox-LDL); Myocardosis; Irregular pulse (inductive after ischemic and the myocardial infarction); Congestive heart failure (CHF); Drug toxicity (comprising adriamycin and Zorubicin); Keshan disease (selenium deficiency); Trypanosomiasis; Alcoholic cardiomyopathy; Venous stasis and damage (comprising venous thrombosis or DVT); Thrombophlebitis;

Lung: asthma; Reactive airway disorders; Chronic obstructive pulmonary disease (COPD or pulmonary emphysema); Hyperoxia; The hyperbaric oxygen effect; Smoke from cigarette sucks effect; Ambient oxidant pollutent effect; Adult respiratory distress syndrome (ARDS); Broncho-pulmonary dysplasia; Mineral pneumoconiosis; Zorubicin toxicity; Bleomycin toxicity; Paraquat 20 and other sterilant toxicity; Chemical pneumonitis; Idiopathic pulmonary interstitial fibrosis; Infectious pneumonia (comprising fungi); Sarcoidosis; Asbestosis; Lung cancer (minicell and maxicell); Anthrax infects; The anthrax toxin contact;

Kidney: hypertensive renal disease, ESRD, diabetic nephropathy, infectious glomerulonephritis, nephrotic syndrome, allergy glomerulonephritis, the allergy of I-IV type, renal homotransplantation repulsion, ephritis property anti-GBM disease, heavy metal renal toxicity, drug-induced (comprising aminoglycoside, furosemide and NSAIDs) renal toxicity, rhabdomyolysis, kidney;

Liver: carbon tetrachloride hepatic injury, intracellular toxin and LPS liver injury, chronic viral infection (comprising virus infection), infectious hepatitis (the non-viral cause of disease), hemochromatosis, hepatolenticular degeneration, PARACETAMOL BP98 are excessive, congestive heart failure companion hepatohemia, liver cirrhosis (comprise alcohol property, a viral and special venereal disease because of), hepatocellular carcinoma, hepatic metastases knurl;

Stomach and intestine: inflammatory bowel (comprising Crohn disease, ulcerative colitis and irritable bowel syndrome), colorectal carcinoma, polyposis, infectious diverticulitis, TMC, gastritis (comprising helicobacter pylori infection), cancer of the stomach, esophagitis (comprising esophageal high grade dysplasia), gastroesophageal reflux disease (GERD), Whipple disease, chololithiasis, pancreatitis, abetalipoproteinemia, gastroenteritis infectiosa, dysentery, NSAIDs inductive toxicity;

Hematopoiesis/blood: Pb (lead) poisons, anaemia, idiopathic thrombocytopenic purpura, autoimmunization deficit syndrome (AIDS) after the drug-induced bone marrow depression, protoporphyrin photoxidation, lymphoma, white blood disease, porphyria, parasitic infection (comprising malaria), sicklemia, thalassemia, favism, pernicious anemia, anemia Fanconi's, infection;

Urogenital: infectious prostatitis, prostate cancer, benign prostatauxe (BPH), urethritis, testitis, testicular torsion, trachelitis, cervical cancer, ovarian cancer, uterus carcinoma, vaginitis, vulvismus;

Muscle skeleton: osteo-arthritis, rheumatoid arthritis, tendinitis, muscular dystrophy, degenerative disc disease, degenerative joint disease, exercise induced skeletal muscle damage, carpal tunnel syndrome, Guillain Barre syndrome, paget's disease of bone, ankylosing spondylitis, dystopy bone forming; With

Body quilt: day irradiation damage (comprising sunburn), thermal damage, chemistry and contact dermatitis (comprising sumach dermatitis), psoriasis, Bloom's syndrome, leukoplasia (particularly mouth), infectious dermatitis, Kaposi.

Second type is many organs illness, and its pathology is associated with radical and non-free radical damage in some aspects convincingly: infringement (comprising Wei-Ke syndrome), ischemia-reperfusion infringement, inflammatory and autoimmune disease, drug toxicity, amyloid disease, overloading syndrome (iron, copper etc.), MSOF and the endotoxemia/sepsis of old and feeble (comprising immunodeficient relevant with the age and too early diseases of aging), cancer, cardiovascular disorder, cerebrovascular disease, radiation injury, alcohol mediation.

Can include but not limited to cardiovascular inflammation, hepatitis C infection, cancer (hepatocellular carcinoma and prostate gland), degeneration of macula, rheumatoid arthritis, apoplexy, Alzheimer with the disease of structural carotenoid analogs treatment, and/or osteo-arthritis.In one embodiment, can suppress and/or improve the generation of treatment target reperfusion injury for the similar thing of treatment target administration of water soluble carrotenoid.In certain embodiments, make up administration of water soluble and other structural carotenoid analogs separately or with other structural carotenoid analogs can for treatment target.Can suppress through water-soluble and/or other structural carotenoid analogs of giving treatment target administering therapeutic amount or improve experience or experienced or tend to experience myocardial infarction, apoplexy, peripheral vascular disease, vein or arterial occlusion, organ transplantation, coronary artery bypass transplantation, through human treatment's object generation reperfusion injury of Pi Jing chamber coronary angioplasty and cardiovascular all standing and/or death.

" water-soluble " structural carotenoid analogs be can be in the aqueous solution separately or those analogues of preparing with vehicle.The water-soluble carotenoid analogue can comprise those compounds and the synthesis of derivatives that forms molecule self-chambering part, and it possibly be called as the similar thing of " water-dispersible " carrotenoid more rightly.Water-soluble and the similar thing of " water-dispersible " carrotenoid possibly be preferred embodiments aspect some in the present invention.

In one embodiment, can suppress for the similar thing of treatment target administration of water soluble carrotenoid and/or the cardiovascular disorder of some type that improvement is relevant with irregular pulse.In certain embodiments, make up the similar thing of administration of water soluble carrotenoid separately or with the similar thing of other carrotenoid can for treatment target.The similar thing of carrotenoid has the elctrical stability that helps keep heart tissue.The help that the heart tissue elctrical stability is kept can suppress and/or improve the cardiovascular disorder of some type, particularly including the sudden cardiac death that is attributable to fatal arrhythmia.

In one embodiment, can suppress and/or improve the generation of treatment target hepatic diseases for the similar thing of treatment target administration of water soluble carrotenoid.In certain embodiments, make up the similar thing of administration of water soluble carrotenoid separately or with the similar thing of other carrotenoid can for treatment target.Said hepatic diseases can be chronic hepatic diseases, for example hepatitis C infection.

In one embodiment, give that the similar thing of treatment target administration of water soluble carrotenoid can suppress and/or improve initially, the propagation and the breeding of conversion and/or cancer cells.In certain embodiments, make up the similar thing of administration of water soluble carrotenoid separately or with the similar thing of other carrotenoid can for treatment target.The similar thing of carrotenoid can suppress the multiplication rate of carcinogens trigger cell.The similar thing of carrotenoid can increase the connection protein 43 expresses.The increase that connects the protein 43 expression can increase, keeps or recover the gap and connect cell-cell communication, thereby suppresses the growth of carcinogens trigger cell.

Embodiment also can relate to the pharmaceutical composition of using the combination that comprises structural carotenoid analogs to this treatment target.A kind of compsn of injectable structural carotenoid analogs-astaxanthin can be used in particular for treat-ment as herein described.In another embodiment; With another kind of astaxanthin analog and/or other structural carotenoid analogs administration, perhaps promote the inhibitor and/or the vehicle of expectation purpose to prepare administration injectable astaxanthin analog with other.In certain embodiments, one or more astaxanthin analogs are water miscible.

In one embodiment, the compound that comprises carrotenoid can have formula (I):

Each R ³Can be hydrogen or methyl independently.R ¹And R ²Can be H independently, have one or more substituent acyclic olefins or comprise one or more substituent rings.In certain embodiments, part is hydrophilic at least for substituting group.These carotenoid derivatives can be used for pharmaceutical composition.In one embodiment, the pharmaceutical composition that comprises the structural carotenoid analogs of formula (I) can be used to treat reperfusion injury.

Term used herein " disodium salt disuccinic acid ester astaxanthin derivatives ", " dAST ", " Cardax ", " Cardax ^TMOn behalf of disodium salt disuccinic acid ester astaxanthin derivatives, ", " rac " and " astaxanthin disuccinic acid ester derivative (ADD) " be used for the different nomenclatures of different steric isomers and water prescription, and the preferred at present but exemplary of representative be used to expect the embodiment of using this structural carotenoid analogs.Diacid disuccinic acid ester astaxanthin derivatives (astaCOOH) is to be used for the verivate of flash photolysis research with the protonated form that directly compares with non-esterified " racemize " (being the mixture of steric isomer) astaxanthin." Cardax-C " is disodium salt disuccinic acid ester two vitamins C verivates (verivate XXIII), is used for superoxide anion and removes experiment, measures through EPR (EPR) imaging.

Brief description of drawings

Combines with accompanying drawing preferred but detailed description that embodiment of the present invention only exemplary are done the at present more fully brief description more than the understanding and further target, the feature and advantage of the inventive method and device with reference to following.

Fig. 1 is the diagram at several kinds of " parent " structural carotenoids of occurring in nature discovery;

Fig. 2 describes with the effect to the reactive oxygen species superoxide anion of the disodium salt disuccinic acid ester astaxanthin derivatives of EPR (EPR) imaging monitoring;

Fig. 3 describes with the effect to the reactive oxygen species superoxide anion of the disodium salt disuccinic acid ester astaxanthin derivatives/free vitamin C solution of EPR (EPR) imaging monitoring;

Fig. 4 describes with disodium salt disuccinic acid ester astaxanthin derivatives iv formulation (Cardax ^TM) the infraction size reduces relatively in the pretreated male Sprague-Dawley rat diagram;

Fig. 5 be described as current research synthetic meso astaxanthin (3R, 3 ' S-or 3S, 3 ' R-dihydroxyl-β, β-Hu Luobusu-4,4 '-diketone; DAST) alltrans is (complete-E) chemical structure of disodium salt disuccinic acid ester derivative (being shown as complete-E two negatively charged ion bolamphiphile);

Fig. 6 describes uv-visible absorption spectra (chamber length 1cm, c=1.05 * 10 of the dAST in ethanol under 25 ℃ ^-5M).Molar absorption coefficient is shown in the bracket.Derive definite position and the vibration fine structure of the main bands of a spectrum of hiding at peak in the nearly UV of curve representation district of second of absorption spectrum;

Fig. 7 describes the Ringer damping fluid, and (pH 7.4, chamber length 1cm, c=1.85 * 10 ^-5M, t=37 ℃) in the absorption spectrum of dAST.Shown molar absorption coefficient;

Fig. 8 describes through being used in inductive CD and the UV/Vis spectrum that the dAST titration human serum albumin (HSA) in the Ringer damping fluid (pH 7.4) obtains with low L/P ratio.The concentration of HSA is 1.6 * 10 ^-4M is the DMSO stock solution (chamber length 1cm, t=37 ℃) of sample aliquot and add part.Shown the curve of under Different L/P value, measuring.Illustration: (Δ ε is with M for the inductive CD that in solution, calculates on the basis of total meso carotenoid concentration and the Mohr Circle of Plastic dichroism uptake factor of absorption band ^-1Cm ^-1Be unit) and molar absorption coefficient (ε is with M ^-1Cm ^-1Be unit);

It is inductive CD and the UV/Vis spectrum that 1 the dAST titration HSA in Ringer damping fluid (pH 7.4) obtains that Fig. 9 describes through using above L/P ratio.The concentration of HSA is 2.3 * 10 ^-4M, and the part that adds is the DMSO stock solution (chamber length 1cm, t=37 ℃) of sample aliquot.Shown that the L/P value is 1.2,2.0,2.9,4.1,5.7 and the curve measured at 7.4 o'clock.CD intensity increases with ligand concentration abreast;

It is inductive CD and the UV/Vis spectrum that 1 the dAST titration HSA in 0.1M pH 7.4 phosphate buffered saline buffers obtains that Figure 10 describes through using above L/P ratio.The concentration of HSA is 2.2 * 10 ^-4M, and the part that adds is the DMSO stock solution (chamber length 1cm, t=37 ℃) of sample aliquot.Shown that the L/P value is 1.2,2.0,2.9,4.1,5.7,9.0,10.6 and the curve measured at 13.1 o'clock.CD intensity increases with ligand concentration abreast;

Figure 11 describes the illustration about the right hand property arrangement of two kinds of meso carrotenoid molecules, in CD spectrum, produces long wave for their exciton interactions and just bears Cotton effect with shortwave.The grey molecule is positioned at the back of paper plane;

Figure 12 describes (last figure): the quenching of fluorescence of the HAS that causes at the dAST that in 0.1M pH 7.4 phosphate buffered saline buffers, measures under 37 ℃.Initial and the ultimate density of HSA and part is respectively 4.2 * 10 ^-6M-4.0 * 10 ^-6M and 1.3 * 10 ^-6M-1.4 * 10 ^-5Change between the M.Mark L/P ratio on curve.(figure below): independent DMSO is to the influence of HSA primary fluorescence.Experiment condition such as text;

Figure 13 describes the X-ray crystal structure of lipid acid-free HSA.Subdomain and two main medicine binding sites of having shown HSA.The bulk in crack between the dotted line representative domain, and the position of Trp214 represented in asterisk.Interatomic distance between 3 and the 3 ' chiral carbon atom of dAST molecule is

Figure 14 describes the statistics mixture (" rac " in the caption) of the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives and induces the functional clearance in the rat embryo fibroblast cell (10T1/2) to connect communication.Handled 4 days like the said culture that will merge of text, measure the ability that shifts optical dye fluorescent yellow then.Arrow representes to inject the cell of fluorescent yellow;

Figure 15 A describes with the connection protein 43 protein expression in the cell of the mixture process of the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives, estimates through quantitative Western engram analysis.Think that top band representative is assembled into the albumen of the phosphorylation form of gap connection; Following band is represented knocked-down albumen (Saez, 1998).1: 1: 2 ethanol (EtOH)/H of swimming lane ₂O (the only negative control of solvent); Swimming lane 2:TTNPB, a kind of synthetic retinoid, in acetone, concentration 10 ^-8M (positive control); Swimming lane 3: retinoic acid ester, in acetone, concentration 10 ^-5M (positive control); Swimming lane 4: the statistics mixture (" rac ") of the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives, concentration 10 ^-5M is 1: 2EtOH/H ₂Send in the O prescription; Swimming lane 5:3R, 3 ' R disodium salt disuccinic acid ester astaxanthin derivatives, concentration 10 ^-5M is 1: 2EtOH/H ₂Send in the O prescription; Swimming lane 6:3S, 3 ' S disodium salt disuccinic acid ester astaxanthin derivatives, concentration 10 ^-5M is 1: 2EtOH/H ₂Send in the O prescription; With swimming lane 7: meso disodium salt disuccinic acid ester astaxanthin derivatives, concentration 10 ^-5M is 1: 2EtOH/H ₂Send in the O prescription;

Figure 15 B describes the immunoblotting with Coomassie blue stain, shows the same protein carrying capacity of all bands.The immune labeled difference of this proof is not to be caused by the variability of loading and/or being transferred to the total protein on the film artificially;

Figure 15 C description and positive control with only compare disodium salt disuccinic acid ester astaxanthin derivatives to being connected the numerical analysis of inducing level relatively of protein 43 protein expression with the contrast of solvent treatment.Swimming lane such as Figure 15 A.To fold induction phase for 1: 2EtOH/H ₂The Cx43 of control level expresses and carries out normalization method in the negative control that O handles, and is set to be A.U.=1.0;

Figure 15 D describes the dose-response curve with Cx43 protein expression in the rat embryo fibroblast cell (10T1/2) of the statistics mixture process of the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives, and it is estimated by quantitative Western engram analysis method.Top band thinks that representative is assembled into the albumen of the phosphorylation form of gap connection; Following band is represented knocked-down albumen.Swimming lane 1: 1: 2EtOH/H ₂O (the only negative control of solvent).Swimming lane 2:TTNPB, in acetone, concentration 10 ^-8M (positive control).Swimming lane 3: disodium salt disuccinic acid ester astaxanthin derivatives (" rac "), concentration 10 ^-5M, the EtOH/H at 1: 2 ₂Send in the O prescription.Swimming lane 4: disodium salt disuccinic acid ester astaxanthin derivatives (" rac "), concentration 5 * 10 ^-6M, the EtOH/H at 1: 2 ₂Send in the O prescription.Swimming lane 5: disodium salt disuccinic acid ester astaxanthin derivatives (" rac "), concentration 10 ^-6M is 1: 2EtOH/H ₂Send in the O prescription;

Figure 15 E description and positive control and the statistics mixture of steric isomer of only comparing disodium salt disuccinic acid ester astaxanthin derivatives with the contrast of solvent treatment are to being connected the numerical analysis of inducing level relatively of protein 43 protein expression.Swimming lane such as Figure 15 D.To fold induction phase for 1: 2EtOH/H ₂The Cx43 of control level expresses and carries out normalization method in the contrast that O handles, and is set to be A.U.=1.0;

The statistics mixture that Figure 16 describes the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives increases the assembling that the Cx43 immunoreactivity connects patch.Statistics mixture like above-mentioned steric isomer with disodium salt disuccinic acid ester astaxanthin derivatives is handled 10T1/2 cytogamy culture 4 days: (1) concentration 10 ^-5M is 1: 2EtOH/H ₂Among the O; (2) with 1: 2EtOH/H ₂The O conduct is the negative control of solvent only; Or (3) TTNPB, concentration 10 ^-8M is in THF (THF) solvent, as positive control.Cellular immunization is dyeed with Cx43 antibody as text is said.A group: the statistics mixture of the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives, concentration 10 ^-5M is 1: 2EtOH/H ₂Among the O; C group: 1: 2EtOH/H ₂O is as solvent control; The E group: TTNPB, concentration is 10 ^-8M is in THF (THF) solvent, as positive control.B, D and F group: respectively A, C and E group are carried out numerical analysis, show that the above pixel of threshold value that fixedly installs is that fluorescence intensity is positive.Yellow arrows: immunoreactivity connects patch; Red arrow: nucleus position.Notice that the quantity and the strength ratio that connect the immunoreactivity patch in the culture with the statistics mixture process of the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives only use the control group of solvent treatment big.C represents patch rare in the control group with the patch that is connected shown in the D group; Most of cell in these cultures is that Cx43 dyeing is negative;

Figure 17 is described as studying at present 4 kinds of steric isomers (being shown as complete-E geometrical isomer) of the disodium disuccinic acid diester of synthetic astaxanthin; The mixture or the independent steric isomer of steric isomer are used for other application of branch (seeing caption);

Figure 18 describes the average inhibition percentage of the disodium disuccinic acid ester derivative of the astaxanthin in the water-based prescription that is detected by DEPMPO spin trap to the superoxide anion signal.The statistics mixture of mixture=steric isomer [3S of 1: 2: 1 ratio, 3 ' S, meso (3R, 3 ' S and 3 ' R, 3S), 3R, 3 ' R].The contrast epr signal (being made as 0% according to convention suppresses) that each verivate in the water prescription is detected when not adding compound carries out stdn.Notice 3S in the water, 3 ' S preparation lacks the restraining effect to super-oxide.In each case, the effectiveness of water prescription is less than the corresponding prescription (Figure 19) in EtOH;

Figure 19 describes the average inhibition percentage of the disodium disuccinic acid ester derivative of the astaxanthin in the ethanol prescription that is detected by DEPMPO spin trap to the superoxide anion signal.The statistics mixture of mixture=steric isomer [3S of 1: 2: 1 ratio, 3 ' S, meso (3R, 3 ' S and 3 ' R, 3S), 3R, 3 ' R].Mixture, meso and 3R, 3 ' R stock solution are 1: 2 ethanol/water (33 ¹/ ₃%EtOH); 3S, 3 ' S stock solution are 1: 1 ethanol/water (50%EtOH).Final EtOH concentration is respectively 0.3% and 0.5% in isolating neutrophil test.The contrast epr signal (being made as 0% according to convention suppresses) that each verivate that will be in the ethanol prescription detects when not adding compound carries out stdn;

The mixture of steric isomer of disodium disuccinic acid ester derivative that Figure 20 describes the astaxanthin that is detected by DEPMPO spin trap to the average inhibition percentage of superoxide anion signal (1: test in the 2EtOH/ water prescription; Final EtOH concentration 0.3% in isolating neutrophil test).Along with the concentration increase of verivate, restraining effect increases with mode non-linear, that depend on dosage.Under 3mM, observe the superoxide anion signal near complete inhibition (95.0% suppresses);

Figure 21 describes the hydrochloride two Methionin astaxanthin derivatives that detected by the DEPMPO spin trap average inhibition percentage to the superoxide anion signal.This verivate height water-soluble (＞50mg/ml), and excellent radical quencher ability does not need solubility promoter in this measures.Superoxide anion restraining effect and Figure 20 of this verivate are compared about the restraining effect shown in the verivate that in the water-based prescription, forms supramolecular assemblies;

Figure 22 describes the standard map of the plasma concns of the afterwards non-esterified free astaxanthin of the single agent per os of black mouse tube feed to the time.In blood plasma, only detect non-esterified free astaxanthin, prove that the similar thing of carrotenoid takes off esterification fully in mammal intestine, as discussed previously;

Figure 23 describes the standard map of the liver concentration of the afterwards non-esterified free astaxanthin of the single agent per os of black mouse tube feed to the time.In liver, only detect non-esterified free astaxanthin, also the similar thing of proof (referring to the Figure 22 about blood plasma) carrotenoid takes off esterification fully in mammal intestine, and is as discussed previously.At each time point, the edema due to dysfunction of the liver of non-esterified free astaxanthin is flat greater than observed level in the blood plasma, and this is a new discovery, shows that the solid organ of dissociation Serlabo in the new emulsion carrier that this institute uses sent to be greatly improved;

Figure 24 describes disodium disuccinic acid ester astaxanthin derivatives through per os tube feed 500mg/kg to the influence of LPS (LPS) inductive mouse liver injury (raise measure through serum alanine aminotransferase or ALT).3 animals of each group test.Salt solution (the false contrast of handling that control animals received is independent; The left part of figure) or do not contain the emulsion (vehicle Control) of disodium disuccinic acid ester astaxanthin derivatives.The background level that the false animals received novel derivative of handling shows ALT does not have influence; The mouse of Orally taken emulsion that acceptance contains the novel derivative of 500mg/kg shows the ALT level of inducing and reduces, and is illustrated in the provide protection of treating liver necrosis after the LPS infringement;

Figure 25 describes with disodium salt disuccinic acid ester astaxanthin derivatives intravenously prescription (Cardax ^TM) the infraction size reduces relatively in the pretreated male Sprague-Dawley rat diagram.Can see dosage and block the linear relationship between big or small the reducing.The infraction size reduces to be on close level to the observed result of local asphyxia pre-treatment;

Figure 26 describes with disodium salt disuccinic acid ester astaxanthin derivatives intravenously prescription (Cardax ^TM) diagram that reduces relatively of infraction size in the pretreated male Sprague-Dawley rat;

Figure 27 describes and uses the transient absorption vs. of the diacid disuccinic acid ester astaxanthin derivatives (astaCOOH) that flash photolysis obtains to postpone.Experiment uses nitronaftalin (NN) to carry out as photosensitizers in acetonitrile (MeCN).The spectrum proof diacid disuccinic acid ester astaxanthin derivatives performance identical with the non-esterified free racemize astaxanthin as the radical quencher (forming the carrotenoid radical cation) of gained identifies that this verivate is for produce the activity " soft medicine " of non-esterified free astaxanthin in vivo after oral and vein are sent;

Figure 28 describes the non-esterified free racemize astaxanthin (asta) of reference compound that uses flash photolysis to obtain] transient absorption vs. postpone.Experiment uses nitronaftalin (NN) to carry out as photosensitizers in acetonitrile (MeCN).The spectrum of gained almost may be superimposed on the spectrum of diacid disuccinic acid ester astaxanthin derivatives (astaCOOH) gained, shows that two kinds of compounds have identical radical-positively charged ion and form performance;

Figure 29 describes the diagram of carrying out polyacrylamide gel Western trace with anti-connection protein 43 antibody;

Figure 30 describes with the anti-protein 43 antibody that connects, and on the Biorad imager, carries out the HRP chemoluminescence then and the diagram of the quantitative image optical density of Western trace that obtains;

Figure 31 be described in take medicine back 96 hours by positive control (TTNPB, effectively synthetic retinoid) and test-compound (disodium salt disuccinic acid ester astaxanthin derivatives is in four kinds of water and/or ethanol (EtOH)/water are filled a prescription: H ₂O-10-5, H ₂O-10-6, H ₂O-10-7 and EtOH/H ₂O-10-5) with respect to sterilized water contrast (H ₂O) to connecting the folding relatively inductive figure that protein 43 is expressed;

Figure 32 is described in the M.L. figure to free astaxanthin non-esterified in blood plasma after the black 500mg/kg disodium disuccinic acid ester astaxanthin derivatives (ADD) of its mouse oral tube feed in emulsion carrier 11 days and the liver.Blood plasma that is reached and the peak value in the liver and valley level all＞200nM, it is considered in vivo oxidative stress and liver injury are had provide protection.The peak level of gained is nearly 9 times (1760nM) of essential protection level in the back 6 hours livers of taking medicine for the 11st time;

Figure 33 describes the disodium salt disuccinic acid ester two vitamins C verivates [verivate (XXIII)] that detected by the DEPMPO spin trap average inhibition percentage to the superoxide anion signal.When the concentration of verivate increased, restraining effect increased with the mode that depends on dosage.Under 60 μ M, observe the superoxide anion signal near the complete inhibition effect.This verivate is water-soluble also very high, and need not solubility promoter and be introduced in the test and (see Figure 21).This novel derivative shows activity, " soft medicine " performance of this verivate aspect radical quencher efficient and with 1: 2 suitable (see figure 3) of prescription with the disodium salt disuccinic acid ester astaxanthin derivatives of vitamins C preparation.This common inhibitor (co-antioxidant) verivate strategy is renderd a service relative free radical scavenging (if comparing with disodium salt disuccinic acid ester astaxanthin derivatives) increases by 50 times;

Figure 34 describes the influence of non-esterified free astaxanthin (as the alltrans mixture of steric isomer) to the tumour conversion of MCA inductive MEC (10T1/2).Non-esterified free astaxanthin produces after oral and intravenous administration novel carotenoid verivate in vivo fast, and in blood and solid organ, detects high density (seeing Figure 22 and 23).Non-esterified free freshwater shrimp element shows tumour and transforms any other carrotenoid that reduction level (100%) surpasses the test in this mensuration of similar concentration, proves that the effectiveness that this compound is used the cancer chemoprophylaxis increases;

Figure 35 describes astaxanthin plate of handling and the comparison (seeing the description about Figure 34) that contrasts plate;

Figure 36 describes astaxanthin (as stereoisomer mixture) and tests the comparison (see description about Figure 34) carried out in this laboratory with this with the carrotenoid of front test;

Figure 37 describes with disodium salt disuccinic acid ester astaxanthin derivatives intravenously prescription (Cardax ^TM) diagram that reduces relatively of infraction size in the pretreated male new zealand rabbit.When in rodent, reducing to compare with same dose and the observed infraction size of identical pretreating scheme, that in rabbit model, observes the infraction size reduces to increase by 38%; With

Figure 38 describes with disodium disuccinic acid ester astaxanthin derivatives intravenously prescription (Cardax ^TM) diagram that reduces relatively of the cyclical level of plasma C-reactive protein (CRP) in the pretreated male new zealand rabbit.Stimulate the contrast rabbit (saline injection separately) that acute phase reaction takes place to show through subcutaneous injection 1% Oleum Tiglii and on average increase by 23.5% with respect to baseline (the vein sample of when pouring into again, being got) circulation CRP level.By contrast, Cardax ^TMThe animal (50mg/kg) of handling shows with respect to baseline circulation CRP level and on average reduces (15.7%), shows Cardax ^TMThe effective antiinflammatory effect.

Detail

" parent " carrotenoid can refer to the natural compounds as the initial skeleton of structural carotenoid analogs synthetic generally.Carotenoid derivatives can be derived from naturally occurring carrotenoid.For example, naturally occurring carrotenoid can comprise Lyeopene, lycophyll, lycoxanthin, astaxanthin, β-Hu Luobusu, xenthophylls, ZXN and/or Food Orange 8.

Carrotenoid is one group of natural pigment that is mainly produced by plant, yeast and microalgae.The quantity of related compound family surpasses 600 kinds of existing members that describe at present, does not comprise Z and E isomer.In human serum or tissue, 50 kinds have been found.People and other animal can not de novo synthesis carrotenoid, must from its diet, obtain them.All carrotenoid has the common chemical feature, and is for example approaching symmetrical around the two keys of cluster isoprene structure, the chromophoric long polyenoid chain of formation and central authorities.Two C ₂₀Tail to the tail of geranyl geranyl bisphosphate molecule connects generation parent C ₄₀Carbon skeleton.The carrotenoid of oxygen-free functional group is called " Serlabo ", reflects their hydrocarbon attribute; Oxidation Serlabo is known as " xenthophylls ".Cyclisation in the one or both ends of molecule produces 7 definite end groups (representative configurations is as shown in Figure 1).

The function of the occurring in nature carrotenoid that confirms comprise light harvesting, light protection and respectively the protectiveness in micro-biology, Mammals and birds with relevant with property painted.Nearer observation is the provide protection of anti-people of carrotenoid and age diseases associated, as the part of the assorted inhibitor network of time multiplexed cell.This effect is given by the plysiochemical performance of each carrotenoid and the close relation between their functions in vivo.The alternately two keys and the single bonded long system (on polyenoid chain total length, making π-orbital electron delocalization) of molecule centre portions give carrotenoid unique shape of molecule, chemical reactivity and absorbing properties.In addition, the isomery around the two keys of C=C produces the visibly different molecular structure that can be separated into independent compound (being known as Z (" cis ") and E (" trans ") or geometrical isomer).In greater than 600 kinds of described carrotenoid, see sometimes even more possible in theory single Z and many Z isomer of more number at occurring in nature.The existing in of the two keys of Z made bigger sterically hindered between contiguous Wasserstoffatoms and/or the methyl, thus Z isomer with corresponding complete-that the E form is compared common thermodynamic stability is less, and chemical reactivity is bigger.Entirely-the E configuration be extend, linearity and stiff molecule.By contrast, Z isomer is not simple linear molecule (so-called " crooked chain " isomer).The existence of any Z produces crooked chain molecule in the polyenoid chain.Z-isomer crystallizes or accumulative trend than complete-E is much little, and Z isomer more dissolves in vivo easily, absorbs and transport than its complete-E counterpart.This is significant for (for example oral) and parenteral in the mammiferous intestines (for example intravenously, intra-arterial, intramuscular and subcutaneous) are taken medicine.

Carrotenoid with chiral centre can be used as R (rectus) or the existence of S (sinister) configuration.For example, astaxanthin (3 and 3 ' carbon have 2 chiral centres) can be used as 4 kinds of possible steric isomers and exist: 3S, 3 ' S; 3R, 3 ' S and 3S, 3 ' R (meso-form); Or 3R, 3 ' R.The relative proportion of each steric isomer possibly become with natural origin.For example, Haematocoocus Pluvialls (Haematococcus pluvialis) microalgae powder is 99%3S, 3 ' S astaxanthin, and possibly be evolve astaxanthin source of dominant people.(3R, 3 ' R) compare with yeast source and little algae source and to produce different steric isomer compsns krill.Synthesizing astaxanthin is produced by giant manufacturer such as Hoffmann-LaRoche AG, Buckton Scott (USA) or BASF AG, with 1: 2: 1 stereoisomer mixture [3S, the 3 ' S of non-esterified free astaxanthin; 3R, 3 ' S, 3 ' R, 3S (meso); 3R, 3 ' R] the geometrical isomer mixture of confirming provide.From the natural origin astaxanthin of salmon flying fish mainly is single stereoisomers (3S, 3 ' S), but comprise the mixture of geometrical isomer.Astaxanthin from natural origin Haematocoocus Pluvialls (Haematococcuspluvialis) can comprise nearly 50%Z isomer.As above-mentioned, the Z conformational change possibly produce higher steric hindrance between two portions of carrotenoid molecule, and its stability is reduced, the reactive increase, and more responsive to reactivity under low oxygen tension.In this case, with respect to entirely-the E form, Z-shaped formula: (1) possibly at first degrade; (2) possibly suppress reactive oxygen species such as superoxide anion attack cells better; (3) possibly preferentially slow down radical forms.In a word, Z-shaped formula possibly favourablely on thermodynamics at first avoided destroying with the lipophilic portion of protecting cell and cytolemma.But; Be important to note that the astaxanthin of complete-E form; Different with β-Hu Luobusu is; With its on β-ionone ring dihydroxyl-with the substituted form of diketone, keep significant oral administration biaavailability and resistance of oxidation, and proved that it has the effectiveness above the increase of β-Hu Luobusu in major part research.The astaxanthin of also inferring complete-E form pair cell in vivo has maximum membrane stabilizing action.Therefore, maybe be in the mixture of natural and synthetic steric isomer the astaxanthin of complete-E form also extremely important in antioxidation mechanism, and possibly be the form that is suitable for the specific medication preparation most.

The antioxidation mechanism of carrotenoid, particularly astaxanthin comprises singlet oxygen quencher, directly free radical scavenging and lipid peroxidation splitting of chain.The polyenoid chain of carrotenoid absorbs the excitation energy of singlet oxygen, shift through stable energy along chain generation delocalization and effectively, and with energy as heat dissipation to local environment.From the energy transfer ratio of triplet state chlorophyll (plant) or other porphyrin and protoporphyrin (in Mammals) to carrotenoid to oxygen with form highly reactive and destructive singlet oxygen ( ¹O ₂) alternative energy shift more easily and take place.Carrotenoid can also be accepted the excitation energy that possibly form in position from singlet oxygen, and once more with energy as heat dissipation to local environment.This singlet oxygen quencher ability has great importance in the generation of cardiac ischemia, degeneration of macula, porphyria and singlet oxygen has other illness of destruction.In the physics quenching mechanism, the carrotenoid molecule can be regenerated (the most common) or lost.The splitting of chain inhibitor that carrotenoid is still excellent, this be a kind of in suppressing lipid peroxidation important mechanism.Astaxanthin can be given unsettled pufas (PUFA) radical contribution hydrogen (H), stops chain reaction.Peroxy radical can also become the immediate cause of MDA chain termination through the polyenoid chain that is added to carrotenoid.The astaxanthin of optimal dose has demonstrated the peroxy radical chain reaction that can suppress fully in the liposome system.Astaxanthin and vitamin E are shared the dual anti-oxidative defense system of this singlet oxygen quencher and direct free radical scavenging, and under most of situation (under the low especially in vivo oxygen tension), are superior to vitamin E as free-radical scavengers and singlet oxygen physics quencher.

Carrotenoid, particularly astaxanthin are effectively directly free-radical scavengers and singlet oxygen quencher, have the character of all expectations of the therapeutical agent that is used to suppress or improves reperfusion injury.Synthetic have " soft medicine " performance (being the activity of derivative form), and the novel carotenoid verivate with cleavable key relevant with the physiology of precursor portions connection can produce the dissociation Serlabo of level of signification in blood plasma and solid organ.Under the situation of non-esterified free astaxanthin, this is a useful especially embodiment (below the non-esterified distinctive characteristic of free astaxanthin):

● crude form liposoluble; Can transform have more to become water-soluble

● molecular weight is 597 dalton [size＜60 dalton (Da) pass hemato encephalic barrier or BBB easily]

● the distinctive long polyenoid chain of carrotenoid, effective in singlet oxygen quencher and lipid peroxidation splitting of chain

● Mammals there is not provitamin A activity (eliminating the too much and toxic misgivings of retinoid of people's vitamin A).

Administration with the inhibitor of direct free-radical scavengers (particularly superoxide anion) should limit hepatic fibrosis through the activation that influences the early stage stellate cells of fiber generation approach and make progress into liver cirrhosis as effective singlet oxygen quencher.Therefore, reduce the ROS level and possibly prevent aspect HSC and the Kupffer Cell activation it is crucial through using good antioxidant.As if this protectiveness antioxygenation extends to the scope of potential therapeutic anti oxygenant, comprises water-soluble (for example vitamins C, gsh, trans-resveratrol) and lipotropy (for example vitamin E, β-Hu Luobusu, astaxanthin) promoting agent.Therefore, water-soluble common inhibitor verivate strategy with the synthetic combination of lipotropy promoting agent is useful especially embodiment.

Generally vitamin E is regarded as with reference to inhibitor.Compare with vitamin E, carrotenoid is the singlet oxygen of quencher in uniform organic solvent and liposome system more effectively.They also are better splitting of chain inhibitor in liposome system.Confirmed that their effects in vivo increase with rendeing a service.They are effective especially under low oxygen tension and lower concentration, and this makes them become special efficacious agents in the disease condition of integral part that local asphyxia is tissue injury and pathology.These carrotenoid also have the natural tropism to liver after oral.Therefore, the treatment administration of carrotenoid should provide the benefit bigger than vitamin E aspect the restriction fibrosis.

Comprise with using some carrotenoid problem relevant: the isomer mixture of the complicacy that (1) provides in natural and synthetic source with structural carotenoid analogs; Comprise non-carrotenoid pollutent, cause increase such as desired security of the mechanism of FDA and the high cost of potency test; (2) limited to treatment target administration artifact availability; (3) difference inducing cell cytochrome p 450 enzyme (this kind of enzyme family shows the specific specificity difference, when zooscopy being extrapolated to the people studying, must consider).

In one embodiment, parent carrotenoid can have the structure of any naturally occurring carrotenoid.Can be as shown in Figure 1 as some instance of the naturally occurring carrotenoid of parent compound.

In certain embodiments, said carotenoid derivatives can comprise the compound with structure (I):

Each R ³Can be hydrogen, methyl, alkyl, alkenyl or aromatic substituent independently.R ¹And R ²Can be independently for H, have at least one substituent acyclic olefin or have at least one substituent ring that has of formula (II):

Wherein n can be between 4-10 carbon atom.W is said substituting group.Substituting group part at least is hydrophilic.Hydrophilic substituent can help to increase the water-soluble of carotenoid derivatives.In certain embodiments, part is water-soluble at least for carotenoid derivatives.Said ring can comprise at least one chiral centre.Said acyclic olefin can comprise at least one chiral centre.Said ring can comprise at least one degree of unsaturation.In some ring embodiment, described ring can be an aromatics.Said ring can comprise substituting group.Substituting group can be hydrophilic.In certain embodiments, said ring can comprise, for example (a) and (b) or (c):

In certain embodiments, substituting group can comprise, for example carboxylic acid, amino acid, ester, alkanol, amine, SULPHOSUCCINIC ACID ESTER, succinate, aminoacetate, ether, glucoside, sugar or carboxylate salt.

In certain embodiments, each substituting group-W can comprise-XR independently.Each X can comprise O, N or S independently.In certain embodiments, each substituting group-W can comprise amino acid, ester, carbamate, acid amides, carbonic ether, alcohol, SULPHOSUCCINIC ACID ESTER or sulphonate independently.In some substituting group embodiment, substituting group can comprise, for example (d)-(pp):

Wherein each R for example is-alkyl-NR independently ¹ ₃ ⁺,-aromatics-NR ¹ ₃ ⁺,-alkyl-CO ₂ ^-,-aromatics-CO ₂ ^-,-amino acid-NH ₃ ⁺,-phosphorylated amino acid-NH ₃ ⁺, polyoxyethylene glycol, Expex, H, alkyl or aryl.In certain embodiments, substituting group can comprise any combination of (d)-(pp).In certain embodiments, electronegative substituting group can comprise basic metal, and a kind of metal or different alkali-metal combination (in having above an electronegative substituent embodiment) are as counter ion.Basic metal can include but not limited to sodium, potassium and/or lithium.

Though the alkene of above structure and structrual description E configuration subsequently, this should not be counted as qualification.The compound that this paper discussed can comprise that alkene is the embodiment of Z configuration or is included in identical intramolecularly Z and the alkene of E configuration combination.Compound as herein described can natural conversion between Z and E configuration, and/or balance exists between two kinds of configurations.

In one embodiment, compound can comprise the carotenoid derivatives with structure (III):

Each Y can be O or H independently ₂Each R can be OR independently ¹Or R ¹

Each R ¹Can be independently-alkyl-NR ² ₃ ⁺,-aromatics-NR ² ₃ ⁺,-alkyl-CO ₂ ^-,-aromatics-CO ₂ ^-,-amino acid-NH ₃ ⁺,-phosphorylated amino acid-NH ₃ ⁺, polyoxyethylene glycol, Expex, H, alkyl, peptide, polylysine or aryl.In addition, each R ²Can be H, alkyl or aryl independently.Said carotenoid derivatives can comprise at least one chiral centre.

In a specific embodiment, wherein Y is H ₂, said carotenoid derivatives has structure (IV)

In a specific embodiment, wherein Y is O, and said carotenoid derivatives has structure (V)

In one embodiment, compound can comprise the carotenoid derivatives with structure (VI)

Each Y can be O or H independently ₂Each R can be H, alkyl or aryl independently.Said carotenoid derivatives can comprise at least one chiral centre.

In a specific embodiment, Y can be H ₂, said carotenoid derivatives has structure (VII)

At Y is that carotenoid derivatives has structure (VIII) in the specific embodiment of O

In one embodiment, compound can comprise the carotenoid derivatives with structure (IX):

Each Y can be O or H independently ₂Each R ' can be CH ₂N can be 1-9.Each X can do independently

Each R can be-alkyl-NR independently ¹ ₃ ⁺,-aromatics-NR ¹ ₃ ⁺,-alkyl-CO ₂ ^-,-aromatics-CO ₂ ^-,-amino acid-NH ₃ ⁺,-phosphorylated amino acid-NH ₃ ⁺, polyoxyethylene glycol, Expex, H, alkyl or aryl.Each R ¹Can be H, alkyl or aryl independently.Said carotenoid derivatives can comprise at least one chiral centre.

At Y is H ₂A specific embodiment in, said carotenoid derivatives has structure (X)

At Y is that said carotenoid derivatives has structure (XI) in the specific embodiment of O

In one embodiment, compound can comprise the carotenoid derivatives with structure (XII),

Each Y can be O or H independently ₂Said carotenoid derivatives can comprise at least one chiral centre.

In a specific embodiment, Y can be H ₂, said carotenoid derivatives has structure (XIII)

At Y is that said carotenoid derivatives has structure (XIV) in the specific embodiment of O

In certain embodiments, compound can comprise the disuccinic acid ester carotenoid derivatives with structure (XV)

In certain embodiments, compound can comprise the disodium salt disuccinic acid ester carotenoid derivatives with structure (XVI)

In certain embodiments, compound can comprise having and the common inhibitor of carrotenoid link coupled (co-antioxidant), the carotenoid derivatives of particularly one or more vitamins C analogues (being the L xitix).Some embodiment can comprise and ascorbic carboxylic acid of carrotenoid link coupled and/or carboxylates derivatives (for example structure (XVII)).

Carbohydr.Res.1978,60, the open oxidation of 251-258 like EQN.5 said vitamin C-6

Some embodiment can comprise and carrotenoid link coupled vitamins C and/or vitamins C analogue.Vitamins C can be through ehter bond and carrotenoid coupling (structure for example

Some embodiment can comprise and carrotenoid link coupled vitamins C disuccinic acid ester analogs (structure (XIX) for example.

Some embodiment can comprise the solution or the pharmaceutical prepn of carrotenoid and/or carotenoid derivatives and common inhibitor, particularly vitamins C and/or the combination of vitamins C analogue.Pharmaceutical prepn can comprise the vitamins C and the carrotenoid of about 2: 1 ratios.

In certain embodiments, can carrotenoid (for example astaxanthin) and vitamins C coupling be formed ehter bond.Ehter bond can be used to react like the described Mitsunobu of EQN.1 and form.

In certain embodiments, vitamins C can the esterification of being selected property.Vitamins C can the being selected property esterification in the C-3 position (for example EQN.2).J.Org.Chem.2000,65,911-913 openly carries out selective esterification with primary alconol in the C-3 position of not protected xitix.

In certain embodiments, can be with carrotenoid and vitamins C coupling.Can be at C-6, C-5 glycol position is coupled to carrotenoid with vitamins C, like EQNS.3 and 4 said, forms acetal.

In certain embodiments, can carrotenoid be coupled to water-soluble portion (for example vitamins C) with the Glyoxylic acid hydrate joint, of EQN.6.Tetrahedron 1989,22, the openly similar acetal formation of 6987-6998

In certain embodiments, can carrotenoid be coupled to water-soluble portion (for example vitamins C) with the Glyoxylic acid hydrate joint, of EQN.7.J.Med.Chem.1988,31, the open Glyoxylic acid hydrate muriate of 1363-1368.

In certain embodiments, can carrotenoid be coupled to water-soluble portion (for example vitamins C) with the SULPHOSUCCINIC ACID ESTER joint, of EQN.8.Carbohydr.Res.1988,176, the open L-xitix 6-SULPHOSUCCINIC ACID ESTER of 73-78.

In certain embodiments, can carrotenoid be coupled to water-soluble portion (for example vitamins C) with the SULPHOSUCCINIC ACID ESTER joint, of EQN.9.Carbohydr.Res.1979,68,313-319 discloses ascorbic 6-bromo derivative.Carbohydr.Res.1988,176,73-78 discloses the reaction of ascorbic 6-bromo derivative and SULPHOSUCCINIC ACID ESTER.

In certain embodiments, can carrotenoid be coupled to water-soluble portion (for example vitamins C) with the SULPHOSUCCINIC ACID ESTER joint, of EQN.10.J.Med Chem.2001,44,1749-1757 and J.Med Chem.2001,44, the open allyl chloride derivative of 3710-3720 and it and nucleophilic reagent comprise the reaction of SULPHOSUCCINIC ACID ESTER under weak basic condition.

In certain embodiments, can carrotenoid be coupled to water-soluble portion (for example vitamins C) with the SULPHOSUCCINIC ACID ESTER joint, of EQN.11.Can carry out selective esterification and vitamins C is coupled to carrotenoid in the C-3 position of not protected xitix with primary alconol.

In certain embodiments, compound can comprise and contains the one or more amino acid (for example Methionin) that are coupled on the carrotenoid and/or the carotenoid derivatives [for example structure (XX)] of amino acid analogue (for example lysine hydrochloride).

In certain embodiments, carotenoid derivatives can comprise:

In one embodiment, said carotenoid derivatives can be synthetic by naturally occurring carrotenoid.Said carrotenoid can comprise the structure 2A-2E described in Fig. 1.In certain embodiments, carotenoid derivatives can be synthetic by comprising the substituent naturally occurring carrotenoid of one or more alcohol.In other embodiments, said carotenoid derivatives can be synthetic by the verivate that comprises the substituent naturally occurring carrotenoid of one or more alcohol.Said synthesizing can produce single stereoisomers.The said synthetic single geometrical isomer that can produce carotenoid derivatives.Said synthesizing/synthesis flow can be included in any purifying formerly or the separating step that carries out on the parent carrotenoid.Instance can include but not limited to 3S, 3 ' S is complete-and the E carotenoid derivatives, wherein parent carrotenoid is astaxanthin.Synthesis flow can comprise to be protected the different functional groups of carrotenoid and/or substituting group precursor, then deprotection.Can alcohol be taken off proton with alkali.Can make alcohol that takes off proton and substituting group precursors reaction with leavings group well.Described alkali can comprise any non-nucleophilic base well known by persons skilled in the art, for example dimethyl aminopyridine.The alcohol that takes off proton can be as nucleophilic reagent and substituting group precursors reaction displacement leavings group.Leavings group can include but not limited to Cl, Br, tosyl group, p-bromobenzenesulfonyl, methylsulfonyl or trifyl.These only are several instances of operable leavings group, and also having mostly is that those skilled in the art are known and conspicuous.In certain embodiments, depend on used leavings group, possibly even will not take off proton by alcohol.In other instance, leavings group can be an intrinsic, and can be included in subsequently in the final structure of carotenoid derivatives, and a non-limiting instance can comprise acid anhydride or strain cyclic ethers.For example, can make the alcohol and the succinyl oxide reaction of taking off proton.In one embodiment, can be further the disuccinic acid ester of astaxanthin be changed into disodium salt.The instance of the synthesis flow of described some particular of preparation partly is described at embodiment.The instance of the following stated is a general limiting examples about the synthesis flow of preparation carotenoid derivatives.

Ischemia-reperfusion (I/R) damage: pathologic, physiologic characteristic

Pour into the local asphyxia cardiac muscle again and cause being in dangerous tissue and tangible cell takes place change, thereby the damage that is produced by the local asphyxia infringement is worsened with local.Particularly, blood vessel and microvascular lesions, endothelial dysfunction, necrocytosis acceleration and granulocyte activation take place in the perfusion back again.

Blood vessel and microvascular lesions are by complement activation; Clq on the cell of circulation and local C-reactive protein and exposure and phosphorylcholine interact and form membrane attack complex (MAC); Necrocytosis and endothelium permeability take place then to be increased, and affected endothelium and activatory white corpuscle produce superoxide anion (O ₂ ^-), microembolus, cytokine release (particularly IL-6), platelet activation is followed the IIbIIIa receptor activation, discharges ADP and serotonin then and causes.Endothelial dysfunction takes place then, and the endothelium by dysfunction produces superoxide anion subsequently, further destroys affected endothelium, forms positive feedback loop.Shown that ischemia-reperfusion causes the early stage and major injury of vascular system, thereby further endangered myocyte's survival.The granulocyte activation is also taking place between flush phase again.The activation of this clone and threshing cause discharging myeloperoxidase (MPO), Pancreatopeptidase E, proteolytic enzyme and oxygen deutero-radical and non-free radical kind (the most important thing is at " respiratory burst " superoxide anion, hypochlorite, singlet oxygen and hydrogen peroxide afterwards).Oxygen deutero-radical and non-free radical (for example singlet oxygen) kind relate to many with local asphyxia with pour into relevant infringement again; And clearly illustrated that lipid peroxidation is again dabbling consequence, measured as forming by thiobarbituricacid active substance (TBARS), mda (MDA) or conjugated diolefine.

Coronary vasodilator endothelium and the damage of myocardium local asphyxia own create help producing can damaging tissue the derive condition of kind of radical and other non-free radical oxygen, it is generically and collectively referred to as reactive oxygen species (" ROS ") at this.People's the xanthine dehydrogenase-xanthine oxidase system based on endothelium is superoxide anion (O ₂ ^-) a source.People's cardiac muscle lacks this kind of enzyme system.In health tissues, 90% enzyme exists as desaturase (D) form; It changes into oxydase (O) form in ischemic tissue.(O)-and form, use molecular oxygen to be electron acceptor(EA), in the coronary artery endothelium, produce superoxide anion O ₂-.Thereby can obtain superoxide anion in local environment, to make other tissue injury.Superoxide anion itself is not the reactive or destructive radical kind of tool in the biosystem.But it is short and long, as to have more destructive radical and/or ROS such as oh group, hydrogen peroxide, singlet oxygen and peroxy radical source of some life-span.Therefore, can think that it is " key " group in the I/R damage.The biological respinse that has below shown the superoxide radical that forms these important oxygenants:

(1) superoxide anion can be accepted single electronics (" monovalence reduction "), produces superoxide (O ₂ ^-2).2 protons of superoxide coupling form hydrogen peroxide (H then ₂O ₂).H ₂O ₂Spread through cytolemma easily, and can not easily from tenuigenin, get rid of, wherein it can react or cascade of active center inflammatory such as nf kappa-B (NF-kappa-B) with cellular component, and said cascade also relates to other inflammatory damage in the I/R damage.

(2) superoxide anion generally self reacts and produces hydrogen peroxide and oxygen (" disproportionation ").Superoxide dismutase can be spontaneous or by enzyme superoxide-dismutase (SOD) catalysis, it is a kind of reaction that causes forming oxidation SOD:

2O ₂ ^-+2H ⁺→H ₂O ₂+ ³O ₂

(3) superoxide anion can be used as reductive agent, and to metallic cation single electronics (" monovalence reduction ") is provided.For example, in following two-step approach, high ferro (Fe ³⁺) be reduced, then as catalyzer with hydrogen peroxide (H ₂O ₂) change into hydroxyl radical free radical (HO ^.).

O ₂ ^-+ Fe ³⁺→ ³O ₂+ Fe ²⁺(step 1)

Then, the ferrous (Fe of reductive metallic cation ²⁺) o-o bond of catalysis fracture hydrogen peroxide.This produces a hydroxyl radical free radical (HO ^.) and a hydroxide ion (HO ^-).Said reaction is known as the Fenton reaction, and it is particularly important in reperfusion injury, wherein iron and/or copper compartmentation forfeiture (generally through red corpuscle RBC haemolysis):

Fe ²⁺+ H ₂O ₂→ Fe ³⁺+ HO ^.+ HO ^-(step 2)

Hydroxyl radical free radical passes cytolemma easily.The hydroxyl radical free radical infringement is " the rate of diffusion restriction ", that is to say, the 3 dimension distances that infringement possibly apply are relevant with the rate of diffusion of group.Hydroxyl radical free radical is a kind of toxic ROS that has especially.Hydroxyl radical free radical can be added to organic substrates (R by in the following reaction representes), and forms this hydroxylation adducts as radical.Under the situation of ischemia-reperfusion injury, the pufas in endothelium and the muscle cell membrane (PUFAs) is responsive especially to the hydroxyl radical free radical infringement:

HO ^.+ R → HOR ^.(hydroxylation adducts)

The adducts that more than forms can also oxidation in the presence of metallic cation or molecular oxygen.This forms the stable product of oxidation.Under first kind of situation, extra transfer transport is to metals ion, and under second kind of situation, is transferred to oxygen (formation super-oxide).Two kinds of adducts radicals can also react each other and form oxidation, stable and crosslinked product and water.This is a significant process in the membranin oxidation:

HOR ^.+HOR ^.→R-R+2H ₂O

In addition, hydroxyl radical free radical can pass through from these molecules, to extract electronics and oxidizing organic substrates:

HO ^.+R→R ^.+OH ^-

Oxidation substrates (R ^.) be radical.These radicals can be in Kettenreaktion and other molecular reaction.

Carrotenoid is especially effectively lipid-peroxo-splitting of chain agent.In one case, the reaction with ground state oxygen produces peroxylradicals (ROO ^.):

R ^.+ ³O ₂→ROO ^-

Peroxylradicals has activity very much.They can react in Kettenreaktion with other organic substrates:

ROO ^.+RH→ROOH+R ^.

Kettenreaktion is common in the oxidative damage of PUFAs and other susceptibility membrane lipid.

The mensuration of oxygen consumption rate is the beginning of Kettenreaktion and an indication carrying out.Be important to note that in the liposome model system oxygen consumption that the non-esterified free astaxanthin of optimal dose can suppress Kettenreaktion fully and follow.

(4) superoxide anion can with hydroxyl radical free radical (HO ^.) reaction formation singlet oxygen ( ¹O ₂*).Singlet oxygen is not a kind of radical, but in the heart biology system, has high activity and destructiveness.Singlet oxygen relate to embrane-associated protein as 5 '-destruction of phosphonuclease, said enzyme has importance (performance is blocked reducing effectively of size to the people) keeping and recover in the partial concn of vasodilation compound such as adenosine:

O ₂ ^-+HO ^.→ ¹O ₂*+HO ^-

(5) superoxide anion can also with free-radical oxidn nitrogen (NO ^.) reaction, produce peroxynitrite (OONO ^-).Peroxynitrite is to have high activity and destructive molecule in the biosystem.

O ₂ ^-+NO ^.→OONO ^-

Polymorphonuclear leukocyte (PMNS), particularly neutrophil and activatory scavenger cell are the abundant sources of oxygen deutero-radical and non-free radical kind.The nadph oxidase system that is arranged in the phagocytic cell cytolemma is the important source that stimulates the back radical.PMNs and activatory scavenger cell consume the oxygen in " respiratory burst " apace, and convert it into superoxide anion, change into hydrogen peroxide (H then ₂O ₂), and a large amount of singlet oxygens.PMNs still is the source of hypochlorite-another kind of destructive reactive oxygen species.Though important in the phagocytic cell activity in infection, in local asphyxia with again in the local environment between flush phase, these ROS attack the normal and impaired host cell in regional areas and further cell injury take place.

Neutrophil is to prolong after the myocardial ischaemia main source of the oxyradical between flush phase again, particularly in the animal model of experimental infraction.Many previous researchs have confirmed during ischemia-reperfusion, to form oxyradical, but the free-radical generating in the myocardial ischaemia that prolongs of seldom having pointed out this radical source or inspected in vivo.Raising in a large number in the formerly ischemic tissue of neutrophil, and be considered to discharge multiple medium through the part, mainly is that oxyradical is induced damage.Before, the activatory neutrophil was still unclear to the contribution of reperfusion injury and potential cardiac muscle rescue.Proposed a kind of method and be used to detect radical, particularly superoxide anion, this method is not disturbed the blood source mechanism of free-radical generating.

Open chest and the dog experience aorta that closes chest and the insertion of coronary artery sinus catheter people such as (, 2001) Duilio.Do not inject chemical substance.Replace, blood is evacuated to the syringe of preparatory filling spin trap, and with EPR (EPR) analysis of spectral method.After inaccessible 90 minutes, the careful concentration of oxyradical is in the back 10 minutes rising several times that flow again, and maintenance obviously raise at least in 1 hour at coronary artery.The radical major part derives from neutrophil, particularly superoxide anion.These radicals show obvious reduction after using following material: the monoclonal antibody (R15.7) of (1) neutrophil NADPH-oxidase inhibitor and (2) anti-neutrophil CD18-adhesion molecule.Design first kind of intervention and be used to reduce the neutrophil respiratory burst, and second kind is used to reduce neutrophil and raises to the reperfusion injury position.Free-radical generating minimizing through monoclonal antibody R15.7 causes is also relevant with obviously less infraction size, and the flow region minimizing is relevant again with the nothing of following.Prove for the first time; The activatory neutrophil is the main source of oxygenant in the perfused hearts again in the body after the time-delay local asphyxia; This phenomenon is permanent the existence, and anti-neutrophil intervention can prevent the increase of the careful concentration of oxyradical between flush phase more effectively.In the animal model of these experimental infractions, the shortage of the pathology that before the coronary artery obturation, is pre-existing in possibly overemphasized neutrophil and raise and the promoter action of activation to the I/R damage; But in fact under the atherosis situation of human artery, the activatory scavenger cell and the activated T lymphocyte that reside in " hazardous location " also can promote the I/R damage greatly.

Local asphyxia causes that ATP exhausts in the involved area cell.Under the plastosome electron transport chain level of the electronics of in common " leakage " about 5% health tissues, processing,, respiratory chain helps further partial information leakage reductive oxygen kind (O particularly when reducing in a large number ₂-).This mainly takes place during local asphyxia.Net effect in the local cells environment is that the redox state equilibrated from anti-oxidant to enzymatic oxidation tilts, and it absorbs additional radical harm and does not cause the ability of further primary cellular defect to reduce simultaneously.

Prevention ischemia-reperfusion injury: the pharmacologically active agents that is used for previous animal and/or people test

In animal model or limited people test, estimate following compound and reduced the therapeutical agent that ischemia-reperfusion injury and/or cardiac muscle rescue during as Acute Myocardial Infarction (AMI).Major part is a biological anti-oxidant.

● superoxide-dismutase (with verivate or stand-in)

● katalase

● gsh and Selenoperoxidase

● xanthine oxidase inhibitor

● vitamins B, C, E (and verivate)

● calcium antagonist

● ACE inhibitor

● sulfydryl mercaptan compound (particularly N-acetylcystein)

● iron chelating agent (DFOM)

● anti-inflammatory agent (for example Ibuprofen BP/EP)

● phosphocreatine

● N-2-mercapto-propionyl-glycin (MPG)

● probucol (and verivate)

● melatonon

● coenzyme q-10

Prove that Acute Myocardial Infarction people patient's endogenous inhibitor exhausts before Singh and India co-worker's the pioneering research, effectively realized that behind AMI cardiac muscle rescues and improves hard traditionally clinical endpoint (for example total cardiac death and non-fatal infraction again) in 30 days and replenish antioxidant blends and/or Coenzyme Q10 99.0 (a kind of effective lipophilic antioxidant) monotherapy.AMISTAD evidence adenosine conduct cardiac muscle in other patient's group of three branches rescues the availability of reagent.RheothRx ^TM(a kind of rheology reagent) also is effective as the rescue agent in people's test, but is abandoned after the renal toxicity secondary.Recently, Medicure, Inc. proof vitamins B verivate in the initial step research of small-sized II of cooperating with Duke ClinicalResearch Institute can be used for cardiac muscle and rescues.Therefore, understand now " translation " problem of recognizing in the review formerly (from the animal model of experimental infraction render a service clinical efficacy) better to the people to the I/R damage.But the coml window of opportunity still exists, because there is not medicament to be rescued reagent by special authorization as human as yet.

The selection of time of myocardial ischaemia-reperfusion injury treatment

As discussed above, pour into the necrocytosis that Acute Myocardial Infarction (mainly adopting pharmacology or operation perfusion again) termination causes owing to local asphyxia in early days again, but cause further damage-most probable to pass through oxidation mechanism contradictorily.People such as Horwitz (1999) have confirmed window of opportunity in 57 dogs, inhibitor must exist to prevent local asphyxia 90 minutes with during pouring into 48 hours more subsequently reperfusion injury taking place with treatment concentration during this period.The statistical study of in this test, carrying out is paid close attention to and is identified the group membership's composition that causes the infraction size differences, and the announcement treatment time length is a main decisive.If in any time of pouring into again in 1st hour, then obviously reduce the infraction size more than or equal to 3 hours infusion.People such as Duilio (2001) oxygen consumption through proof reflection peroxylradicals chain reaction in canine model was keeping raising further this problem of clarification at least in dabbling 11 hour again pouring into beginning in back 10 minutes and free radical activity again.People such as Singh (1996) before proved in people patient, average 13 hours initial antioxidant therapies behind MI, and continue 28 days, and then to rescue and improve hard clinical endpoint (non-fatal infraction again, death) be possible to cardiac muscle.Therefore, the plasma antioxidants the long half-lift of having possibly be particularly suitable for this situation, because they can be with the loading dose administration, and decay in blood plasma early stage (0-24 hour) behind the AMI of whole key.Scope plasma half-life 222 hours of oral carrotenoid from about 21 hours of xenthophylls (" oxidation " carrotenoid comprises astaxanthin, Capsanthin, xenthophylls and ZXN) to Serlabo (" hydrocarbon " carrotenoid such as Lyeopene).In the test of people such as Horwitz (1999) superoxide-dismutase and stand-in thereof the plasma antioxidants transformation period (7 minutes) in people research with about nearly 21 hours of xenthophylls with given prominence to the pharmacokinetics advantage of carrotenoid in people AMI and the potential cardioprotection of antagonism I/R damage about the significant difference of nearly 9 days transformation period of Serlabo.

The key evaluation of inhibitor in reperfusion injury: people's research

In the research that people such as Singh (1994) carry out, vitamin A, C, E and the β-Hu Luobusu M.L. of comparing the patient of performance AMI with control patients obviously reduce.AMI patient's MDA obviously raises.Smoking and mellitus that negative pass between AMI and the low VITAMINs blood plasma level ties up to these patients of adjusting are afterwards still obvious.Similarly, people such as Levy (1998) have studied 38 AMI patients, and they compare with the normal healthy controls object that the age is complementary and show vitamin A, E and β-Hu Luobusu level and obviously reduce.After thrombolysis, the lipid peroxidation product among the treatment patients serum obviously raises.Thromboembolism treatment also causes the plasma vitamin E level obviously to reduce.The antioxidant vitamin serum level that the research of these descriptions is illustrated in the patient of experience acute coronary event when presenting AMI possibly reduce.Under acute situations, carry out pharmacological intervention and may correct antioxidant vitamin shortage and whole body antioxidant status with anti-oxidant compounds.

Under the situation of elementary and/or secondary prevention CVD, carrying out perspective people with inhibitor, to intervene test limited equally, but great majority are successful.4/5 present people studies the validity of strong support vitamin E aspect minimizing heart trouble risk and complication rate.The inhibitor that the patient who suffers from obvious ephrosis is carried out discloses the secondary prevention research of the cardiovascular disorder in the end-stage renal disease, and patient's the non-lethal MI that gives 800IU/ days natural origin vitamin E reduces by 70%.Similarly, like what this paper mentioned, the cardiac muscle that many medicaments have been successfully used to the people at present rescues application.

Intravenously is sent low-molecular weight compound to suppress or to improve I/R and damage its immunogenicity of needs assessment under acute situations.Blood transfusion type to the fast infusion compound should minimize with other incidence rate of adverse reaction.The compound of molecular weight＜1000Da, for example Frosst), progesterone and astaxanthin possibly not have immunogenicity, only if compound with carrier.When molecular weight is increased to 1000-6000Da, for example Regular Insulin and ACTH, compound possibly be or possibly not be immunogenic.When molecular weight increase to＞during 6000Da, compound possibly be immunogenic.In addition, lipid seldom has immunogenicity, only if same compound with carrier.Astaxanthin, as a kind of xenthophylls carrotenoid, its crude form has highly fat-soluble.Its size also little (597Da).Therefore, the immunogenicity possibility of injectable astaxanthin analog in appropriate prescription is low, is the special ideal compound that is used for treating at present indication.

Prevention irregular pulse: be used for previous zooperal pharmacologically active agents

The mouse of the research evaluation genetic modification that people such as Gutstein (2001) carry out, it can not be expressed in cardiac muscle and connect protein 43 [the Cx43 condition knocks out (CKO) mouse].Mouse has normal heart structure and shrinkage although people such as Gutstein find Cx43 CKO, and sudden cardiac death as one man takes place for they, obviously is because spontaneous mortality ventricular tachycardia.These data support the gap connecting passage particularly to connect the keying action of protein 43 in keeping cardiac electric stability.Can connect the connection albumen that protein 43 is wide expression in people's tissue by the carrotenoid inductive.Therefore, carrotenoid and structural carotenoid analogs can be used to treat irregular pulse.

Preventing cancer: be used for previous zooperal pharmacologically active agents

Mainly in animal model, estimate the possible therapeutic value of carrotenoid aspect prevention and treatment cancer.Before, the antioxidant property of carrotenoid related to the focus of the research of carrotenoid and the application in cancer prevention thereof.As if people such as Bertram (1991) are though carrotenoid is pointed out in the research of carrying out is inhibitor, and this specific performance properties is not to cause their active principal elements as cancer chemopreventive agent.But the activity of finding carrotenoid is very relevant with the ability of their rise gap junction communications.Infer the gap and connected the transfer canal that serves as the antiproliferative signal that produces by the downtrod normal cell of growth.Can connect the connection albumen that protein 43 is wide expression during the people organizes by the carrotenoid inductive.Therefore, raising the connection protein 43 possibly be the mechanism that carrotenoid is used for the cancer of chemoprophylaxis people and other animal.And recently the people that carries out of people (2003) such as Nishino study the carrotenoid mixture of proof through long-term orally give (the 10mg Lyeopene, α-with each 5mg of β-Hu Luobusu) hepatocellular carcinoma of the high-risk liver cirrhosis patient of chemoprophylaxis Japan effectively.The carrotenoid (for example astaxanthin) of the chemoprophylaxis cancer of therefore in liver, accumulating more significantly with bigger effectiveness possibly be useful especially embodiment.Carrotenoid is used to treat ischemia-reperfusion injury, hepatic diseases, irregular pulse and cancer

The application of disease

Term used herein " inhibition " and " improvement " are commonly defined as and prevent and/or the negative results of the state that palliates a disease.Therefore, method and composition as herein described possibly all have value as acute and chronic (preventative) form.

Term used herein " ischemia-reperfusion injury " is commonly defined as owing to the pathologic condition that reoxidizes to previous ischemic tissue (chronic or acute ischemic), and it comprises atherosclerosis and thromboembolic states vascular disease and relevant disease thereof.Particularly; Main disease or process comprise myocardial infarction, apoplexy, peripheral vascular disease, vein or arterial occlusion, organ transplantation, coronary artery bypass transplantation, are included through Pi Jing chamber coronary angioplasty and cardiovascular all standing and/or death, but they are not considered to be in its pathology separately, relating to the ischemic tissue restriction of dabbling other pathologic process again.

Term used herein " irregular pulse " is commonly defined as any variation of the normal rhythm that is different from heartbeat, comprises that sinus arrhythmia, premature beat, heart-block, atrial fibrillation, room are pounced on, ventricular tachycardia, chamber quiver, diapulse and paroxysmal tachycardia.Term used herein " irregular pulse " is commonly defined as the disorder of cardiac electrical activity, and it shows as the unusual of the heart rate or the rhythm of the heart.The most common and the cardiovascular disorder of irregular pulse, particularly ischemic heart disease is relevant.

Term used herein " cancer " it is generally acknowledged and it is characterized in that not controlled, unusual cell growth.Particularly, cancer possibly refer to diseased tissue, comprises cell and carcinogens cell transformed that carcinogens causes.

Term used herein " structural carotenoid analogs " can be commonly defined as carrotenoid and biologically active structure analogue thereof.Typical analogue comprises biological useful with the relevant function that performance is equal or improve, but structurally is different from the molecule of parent compound.

Parent carrotenoid be selected from described in the document more than 600 kinds of naturally occurring carrotenoid and their solid and geometrical isomer.These analogues can include but not limited to ester, ether, carbonic ether, acid amides, carbamate, SULPHOSUCCINIC ACID ESTER and ether, sulfuric ester, glucosides ether, contain or do not contain (joint) at interval.

Term used herein " more than a kind of synergistic combination of structural carotenoid analogs " can be commonly defined as any compsn that comprises a kind of structural carotenoid analogs and one or more other structural carotenoid analogs or common antioxidant combination (as verivate or in solution and/or preparation).

Term used herein " treatment target " can be commonly defined as all Mammalss, particularly people.

Term used herein " administration " can General Definition through method drug administration or the nonprescription drugs (OTC) or the nutritive compsns of any realization expectation target.For example, administration can comprise in parenteral, subcutaneous, intravenously, the coronary artery, rectum, intramuscular, intraperitoneal, transdermal or contain the clothes approach.Can select or side by side, administration can by oral route.Dosage depends on recipient's age, health, body weight and morbid state, the type (if any) of treating simultaneously, the character of therapeutic frequency and target effect.Any technology that is intended to suppress ischemia-reperfusion injury as herein described also can be used for suppressing or improving hepatic diseases, and limiting examples is a hepatitis C infection.The technology that suppresses and/or improve ischemia-reperfusion injury that relates to as herein described also can be used for suppressing and/or improving irregular pulse.The technology that suppresses and/or improve ischemia-reperfusion injury that relates to as herein described also can be used for suppressing and/or improving cancer.

An embodiment can comprise to the independent or co-administered structural carotenoid analogs of treatment target, thereby suppresses and/or improve the generation of ischemia-reperfusion injury.Said structural carotenoid analogs can be water-soluble and/or the water-dispersible verivate.Said carotenoid derivatives can comprise the water miscible any substituting group that increases naturally occurring carrotenoid in fact.

Said carotenoid derivatives can keep and/or improve the antioxidant property of parent carrotenoid.Said carotenoid derivatives can keep the nontoxic performance of parent carrotenoid.Said carotenoid derivatives can have the bioavailability of raising with respect to parent carrotenoid to the treatment target administration time.Said parent carrotenoid can naturally exist.

Another embodiment can comprise to treatment target uses the compsn that comprises more than a kind of synergistic combination of structural carotenoid analogs, reduces the generation of ischemia-reperfusion injury whereby.Said compsn can be carotenoid derivatives " racemic " (being the mixture of a potential stereoisomeric forms in any ratio) mixture.Also comprise the pharmaceutical composition that contains the structural carotenoid analogs that makes up with pharmaceutically acceptable carrier (for example human serum albumin).In one embodiment, can the analog and the human serum albumin (being HSA) of carrotenoid is compound in solvent.HSA can be used as pharmaceutically acceptable carrier.

In certain embodiments; Compsn can comprise all compsns of 1.0 grams or specific structural carotenoid analogs still less and 1.0 grams or one or more other structural carotenoid analogs still less and/or common antioxidant combination, and its amount effectively realizes the purpose of expectation.Though the individual treatment object need variation, the optimum range of the significant quantity of each component fixes in the skill of this area really.Typically, structural carotenoid analogs can be applied to Mammals, particularly people, and with reference to being treated the Mammals of ischemia-reperfusion injury or people's body weight, oral dosage is 5-100mg/ days.Typically, can structural carotenoid analogs be applied to Mammals, particularly the people treats the Mammals of reperfusion injury or people's body weight with reference to quilt, and administered parenterally dosage is 5-500mg/ days.In other embodiments, the about 100mg structural carotenoid analogs of oral or parenteral administration is with treatment or prevention ischemia-reperfusion injury.

The unit oral dosage can comprise the structural carotenoid analogs of about 0.25mg to about 1.0 grams or about 5-25mg.Unit parenteral dosage can comprise the structural carotenoid analogs of about 25mg-1.0 gram or 25mg-500mg.Dosage can comprise the structural carotenoid analogs of about 25mg-1.0 gram or 25mg-100mg in the unit coronary artery.Unitary dose can be administered once or repeatedly every day, administration every other day, and with loading dose or bolus form administration, perhaps titration substitutes mark or clinical endpoint to conventional that accept or new biological chemistry in parenteral solution, and these belong to the technical ability of this area.

Except structural carotenoid analogs is used as the raw material chemical substance, can be with a part of administration of compound as the pharmaceutical prepn that includes suitable pharmaceutically acceptable carrier, sanitas, vehicle and the auxiliary agent that are beneficial to pharmaceutically useful structural carotenoid analogs processing.Preparation; Particularly can be oral and can be used for the preparation of the administration of preferred type; For example tablet, soft gelatin, lozenge, lozenge and capsule, and preparation that can rectal administration, for example suppository; Be used to inject or the suitable solution of oral administration, can as the above-mentioned dosage range that similar bioavailability is provided in prepare with vehicle.

Pharmaceutical prepn can mode well known by persons skilled in the art prepare, for example through conventional mix, granulate, sugaring ingot, soft gelatin encapsulated, dissolving, extraction or freeze drying process.Therefore, medicine preparation for oral use can obtain through active compound and solid and semisolid excipient and suitable sanitas and/or common inhibitor are merged.Randomly, can the mixture of gained be ground and processing.If desired or essential, can be after adding suitable auxiliary agent the particulate mixture of gained be used to obtain tablet, soft gelatin, lozenge, capsule or lozenge core.

Suitable vehicle can be a weighting agent, carbohydrate (for example lactose, sucrose or seminose) for example, sugar alcohol (for example N.F,USP MANNITOL or sorbyl alcohol), cellulose preparation and/or calcium phosphate (for example tricalcium phosphate or secondary calcium phosphate).In addition; Can use tackiness agent, for example starch paste (for example Zea mays or W-Gum, wheat starch, rice starch, yam starch, gelatin, tragacanth, methylcellulose gum, Vltra tears, Xylo-Mucine and/or Vinylpyrrolidone polymer).Can add disintegrating agent (for example above-mentioned starch) and CMS, cross-linked polyvinylpyrrolidone, agar or alginic acid or its salt (for example sodiun alginate).Auxiliary agent at first is flowing regulator and lubricant (for example silicon-dioxide, talcum, Triple Pressed Stearic Acid or its salt, for example Magnesium Stearate or calcium stearate, and/or polyoxyethylene glycol or PEG).The lozenge core provides with suitable dressing, if necessary its tolerance gastric juice.Soft gelatin capsule (" soft gelatin ") provides suitable dressing, said dressing generally to comprise gelatin and/or suitable edible dyestuff.Do not contain animal ingredient and edible gelatine capsule because use widely and consume and possibly be specially adapted to embodiment as herein described.For this purpose; Can use spissated sugar soln; It can be chosen wantonly and comprise gum arabic, talcum, Vinylpyrrolidone polymer, polyoxyethylene glycol (PEG) and/or titanium oxide, lacquer solution and suitable organic solvent or solvent mixture, comprises methyl-sulphoxide (DMSO), THF (THF), acetone, ethanol or other The suitable solvent and solubility promoter.In order to produce the dressing of resistant to gastric juice, can use the for example solution of cellulose acetate phthalic ester or hydroxypropylmethylcellulose phthalate of suitable cellulose preparation.Can dyestuff or pigment be added to tablet or lozenge dressing or soft gelatin capsule, for example be used to discern and, perhaps cover up capsule 's content and be used for clinical or other research in order to characterize the combination of active compound doses.

Other medicines preparation that can mouthful usefulness comprises by the pushing of gelatin preparation-capsule-containing and the soft heat-sealing capsule that prepared by gelatin and softening agent such as glycerine or sorbyl alcohol.Push-capsule-containing can comprise the active compound of particle form, its can with weighting agent such as lactose, tackiness agent such as starch and/or lubricant such as talcum or Magnesium Stearate, and randomly stablizer and/or sanitas mix.In soft capsule, can active compound be dissolved or suspended in appropriate liquid, for example in wax such as Rice pollard oil or peanut oil or plam oil or the whiteruss.In other embodiments, can add stablizer and sanitas.

The possible pharmaceutical prepn that can rectum uses for example comprises the suppository of being made up of the combination of active compound and suppository base.For example suitable suppository base is natural or synthetic glycerine three esters or paraffinic hydrocarbons.In addition, can also use the gelatin rectal capsule of forming by the combination of activeconstituents and matrix.Possible substrate material for example comprises liquid triglycerides, polyoxyethylene glycol or paraffinic hydrocarbons.

The suitable prescription that is used for administered parenterally includes but not limited to the aqueous solution of the active compound of water-soluble and/or water-dispersible form, for example water-soluble salt, ester, carbonic ether, SULPHOSUCCINIC ACID ESTER or ether, sulfuric ester, glucosides ether, and interval and/or joint.In addition, can use suspension-s, be particularly suitable for intramuscularly as the active compound of suitable oily injectable suspensions.Can use suitable lipophilic solvent, solubility promoter (for example DMSO or ethanol), or vehicle comprise wax such as Rice pollard oil or peanut oil and/or plam oil or Acrawax such as OE or triglyceride level.Water injection suspension liquid can comprise the material that increases suspension viscosity, for example comprises Xylo-Mucine, sorbyl alcohol, Expex and/or Schardinger dextrins.(for example (beta-cyclodextrin) can specifically be used to increase and be used for the water-soluble of parenteral injection structural carotenoid analogs Schardinger dextrins.Can prepare and comprise structural carotenoid analogs and the for example liposome prescription of the mixture of lecithin phatidylcholine (E-PC) and be used for injection.Randomly, said suspension-s can also comprise stablizer, for example inhibitor such as BHT or sanitas such as benzylalcohol.

Embodiment

Described the present invention now, be more readily understood the present invention with reference to following examples, said embodiment only provides with illustrative mode, and is not intended to qualification the present invention.

About the synthetic and sign of compound described herein, reagent uses available from commercial source and former state, only if point out in addition.Being used for reaction and isolating solvent is the not purified use of SILVER REAGENT, only if point out in addition.Be reflected at nitrogen (N below all ₂) carry out under the atmosphere, and avoid direct light." racemize " astaxanthin (being the steric isomer 3S of 1: 2: 1 ratio, 3 ' S, meso and 3R, the mixture of 3 ' R) is available from Divi ' s Laboratories, and Ltd (Buckton Scott, India)." racemize " xenthophylls and ZXN are available from Indofine Chemical Co., Inc.Carry out thin-layer chromatography (TLC) with Uniplate silica gel G F 250 micron plates.The HPLC that is used for process control (IPC) analyzes and carries out with Varian Prostar Series 210 liquid chromatographs; This chromatographic instrument has Alltech Rocket; Platinum-C18,

, 3 μ m; 7 * 53mm, PN 50523; Temperature: 25 ℃; Moving phase: (A=water; The B=10% methylene chloride), 40%A/60%B (initial); The internal linear gradient was to 100%B in 8 minutes; Kept 100%B 4 minutes, the internal linear gradient was to 40%A/60%B in 1 minute; Flow velocity: 2.5mL/ branch; Initial pressure: 2050PSI; PDA detector wavelength: 474nm.Write down NMR with Bruker Advance 300, and carry out mass spectroscopy with ThermoFinnigan AQA spectrograph.With Agilent1100LC/MSD VL ESI system log (SYSLOG) LC/MS; Post: Zorbax Eclipse XDB-C18Rapid Resolution (4.6 * 75mm, 3.5 μ m, USUT002736); Temperature: 25 ℃; Initial pressure: 107 crust; Flow velocity: 1.0ml/ branch; (%A=0.025%TFA is at H for moving phase ₂Among the O, %B=0.025%TFA is in acetonitrile) method 1 (compound 8-21,23-27; 30,31): 70%A/30%B (initially), the sublevel gradient is to 50%B in 5 minutes; 8.30 the sublevel gradient kept 15.20 minutes at 98%B to 98%B in minute, 15.40 interior sublevel gradients are to 30%B; Method 2 (compound 28,29): 70%A/30%B (initially), the sublevel gradient is to 50%B in 4 minutes; 7.30 the sublevel gradient is to 90%B in minute; 10.30 the sublevel gradient remained on 98%B 15.20 minutes to 98%B in minute, the sublevel gradient is to 30%B in 15.40 minutes; Method 3 (compound 22): 70%A/30%B (initially), the sublevel gradient is to 50%B in 5 minutes, and the sublevel gradient kept 25.20 minutes at 98%B to 98%B in 8.30 minutes, and the sublevel gradient is to 30%B in 25.40 minutes; PDA detector: 470nm; The LRMS:+ pattern, ESI.

Astaxanthin (2E) .HPLC RT: 11.629 minutes, 91.02% (AUC); LRMS (ESI) m/z (relative intensity): 598 (M ⁺+ 2H) (60), 597 (M ⁺+ H) (100); The HPLC RT: 12.601 minutes, 3.67% (AUC); LRMS (ESI) m/z (relative intensity): 597 (M ⁺+ H) (100); The HPLC RT: 12.822 minutes, 5.31% (AUC); LRMS (ESI) m/z (relative intensity): 597 (M ⁺+ H) (100).

Xenthophylls (XXX) .HPLC RT: 12.606 minutes, 100% (AUC); LRMS (ESI) m/z (relative intensity): 568 (M ⁺) (100).

ZXN (XXXI) .HPLC RT: 12.741 minutes, 100% (AUC); LRMS (ESI) m/z (relative intensity): 568 (M ⁺) (100).

Embodiment 1: synthetic XV (the disuccinic acid ester of astaxanthin (succsinic acid one-(4-{18-[4-(3-carboxyl-propionyloxy)-2,6,6-trimethylammonium-3-oxo-hexamethylene-1-thiazolinyl]-3,7; 12,16-tetramethyl--octadecane-1,3,5; 7,9,11,13; 15,17-nonene base }-3,5,5-trimethylammonium-2-oxo-hexamethylene-3-thiazolinyl) ester))

Under the room temperature toward at the astaxanthin 2E (6.0g of DCM (" methylene dichloride ") in (50mL); 10.05mmo solution l) adds DIPEA (" N; N-diisopropyl ethyl amine ") (35.012mL; 201mmo l), succinyl oxide (10.057g, 100.5mmol) and DMAP (" 4-(dimethylamino) pyridine ") (0.6145g, 5.03mmol).Reaction mixture was at room temperature stirred 48 hours, and, with salt solution/1M HCl (60mL/10mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.Use Na ₂SO ₄The dry organic layer that merges, and the concentrated astaxanthin disuccinic acid ester (XV) (100%) that obtains.The HPLC RT: 10.031 minutes, 82.57% (AUC); LRMS (ESI) m/z (relative intensity): 798 (M ⁺+ 2H) (52), 797 (M ⁺+ H) (100); The HPLC RT: 10.595 minutes, 4.14% (AUC); LRMS (ESI) m/z (relative intensity): 797 (M ⁺+ H) (40), 697 (100); The HPLC RT: 10.966 minutes, 5.68% (AUC); LRMS (ESI) m/z (relative intensity): 797 (M ⁺+ H) (100), 679 (31); The HPLC RT: 11.163 minutes, 7.61% (AUC); LRMS (ESI) M/Z (relative intensity): 797 (M ⁺+ H) (38), 679 (100), and do not have detectable astaxanthin 2E.

Embodiment 2: synthetic XVI (the disuccinic acid ester of astaxanthin (succsinic acid one-(4-{18-[4-(3-carboxyl-propionyloxy)-2,6,6-trimethylammonium-3-oxo-hexamethylene-1-thiazolinyl]-3,7; 12,16-tetramethyl--octadecane-1,3,5; 7,9,11,13; 15,17-nonene base }-3,5,5-trimethylammonium-2-oxo-hexamethylene-3-thiazolinyl) ester) disodium salt)

(2g 2.509mmol) is stirring in the 500mL round-bottomed flask under room temperature and the nitrogen atmosphere with 200mL ethanol with the disuccinic acid ester of astaxanthin.Disposable adding sodium ethylate (340mg, 5.019mmol, Acro s#A012556101) solid, and solution stirring spent the night.Second day, leach deposition, use washing with alcohol, obtain a kind of purple solid with washed with dichloromethane then, the disodium salt of the disuccinic acid ester of astaxanthin, XVI [1.41g, 67%] is placed in the high vacuum pipeline dry. ¹H-NMR (the δ 6.77-6.28 of methyl alcohol-d4) (14H, m), 5.53 (2H, dd, J=12.6,6.8), 2.68-2.47 (8H, m), 2.08-1.88 (22H, m), 1.37 (6H, s), 1.24 (6H, s); ¹³C NMR (CDCl ₃) δ 196.66,180.80,175.01,163.69,144.12,141.38,138.27,136.85; 136.12,135.43,132.35,129.45,126.22,124.71,72.68,44.09; 38.63,34.02,32.34,31.19,26.86,14.06,13.19,12.91; Mass spectrum+ESI, 819.43 1 sodium salts, the disuccinic acid ester of 797.62 astaxanthins; HPLC 7.41 minutes (99.84%).

Embodiment 3: the BocLys of synthesizing astaxanthin (Boc) OH ester (XXI).

HPLC: post: 3.5 microns 4.6mm * 150mm of Waters Symmetry C18; Temperature: 25 ℃; Moving phase: (A=0.025%TFA is at H ₂Among the O; B=0.025%TFA is in MeCN), 95%A/5%B (initial); Linear gradient to 100%B experience 12 minutes kept 4 minutes; Linear gradient to 95%B/5%A experience 2 minutes; Linear gradient to 95%A/5%B experience 4 minutes; Flow velocity: 2.5ml/ branch; Detector wavelength: 474nm.

Toward the astaxanthin 2E (11.5g in methylene dichloride (500mL); 19.3mmol) and BocLys (Boc) OH (20.0g, (10.6g is 86.6mmol) with 1 for mixture adding 4-dimethylaminopyridine (DMAP) 57.7mmol); 3-di-isopropyl carbodiimide (" DIC ") (13.4g, 86.7mmol).Cover round-bottomed flask with aluminium foil, and with mixture stirred overnight under room temperature and nitrogen atmosphere.After 16 hours, incomplete according to HPLC and TLC reaction.Other 1.5 normal DMAP and DIC are added to reactant, react completely according to HPLC after 2 hours.Then mixture is concentrated into 100mL, leaches a kind of white solid (1,3-di-isopropyl urea).(10%-50% heptane/EtOAc) filtrating is carried out the flash chromatography processing to obtain title product is a kind of garnet solid (XXI) (28.2g,＞100% productive rate) with silica gel. ¹H NMR (DMSO-d ₆) δ 7.24 (2H, t, J=6.3Hz), 6.78 (2H, d, 5.0Hz), 6.57-6.27 (14H, m), 5.50-5.41 (2H; M), 3.99-3.97 (2H, d, 6.0Hz), 2.90 (4H, m), 2.03 (4H, m); 2.00 (6H, s), 1.97 (6H, s), 1.82 (6H, s), 1.70-1.55 (4H, m); 1.39-1.33 (36H, m), 1.24-1.13 (8H, m), 1.01-0.99 (6H, m), 0.86-0.83 (6H, m) .HPL:21.3 minute (24.6%AUC)); Minute 22.0 (48.1% (AUC)); 22.8 minute (20.6% (AUC)) .TLC (1: 1 heptane/EtOAc:R _f0.41; R _f0.5; R _f0.56). through the holotype flow injection at Agilent 1100LC/MSD

Carrying out LC/MS in the VL ESI system analyzes; Moving phase: A=is at H ₂0.025%TFA among the O; The 0.025%TFA of B=in MeCN, 10%A/90%B (initial); Initial pressure: 10 crust; PDA detector 470nm.+ESI, m/z=1276.1 (M+Na ⁺).

Embodiment 4: four hydrochlorides (XX) of two Methionin esters of synthesizing astaxanthin.

With DiBocLys (Boc) ester (XXI) of astaxanthin (20.0g, 16.0mmol) with 1, the HCl in the 4-diox (4.00M, 400mL, 1.60mol, mixture 100eq) stirs under room temperature and nitrogen atmosphere.Cover round-bottomed flask with aluminium foil, and reactant was stirred 1 hour, react completely according to HPLC this moment.The deposition title compound is collected with filtration method, with ether (3 * 100mL) washings, and drying (14.7g, 92%, be 91.6% purity according to HPLC).The thick solid of part (13.5g) is dissolved in 1: 2 ethanol/methylene mixture of 500mL, and under nitrogen atmosphere, stirs.Drip ether (168mL) then, and obtain title product, be a kind of garnet solid (8.60g, 63.7% productive rate) with the solid of filtration method collecting precipitation. ¹HNMR (DMSO-d ₆) δ 8.65 (6H, s), 8.02 (6H, s), 6.78-6.30 (14H, m), 5.59-5.51 (2H; M), 4.08 (2H, m), 2.77 (4H, m), 2.09-2.07 (4H, m); 2.01 (6H, s), 1.97 (6H, s), 1.90-1.86 (4H, m), 1.84 (6H; S), 1.61-1.58 (8H, m), 1.37 (6H, s), 1.22 (6H, s) .HPLC:7.8 minute (97.0% (AUC)).Carry out LC/MS with Agilent 1100LC/MSD VL ESI system and analyze, said system has Zorbax Eclipse XDB-C18 Rapid Resolution4.6 * 75mm, 3.5 microns, USUT002736; Temperature: 25 ℃; Moving phase: (%A=0.025%TFA is at H ₂Among the O; %B=0.025%TFA is in MeCN), 70%A/30%B (initial); Linear gradient experiences 5 minutes to 50%B, and linear gradient experiences 7 minutes to 100%B; Flow velocity: 1.0mL/ minute; Initial pressure: 108 crust; PDA detector 470nm.Mass spectrum+ESI, m/z=853.9 (M+H ⁺), m/z=875.8 (M+NA ⁺); LC 4.5 minutes.

Embodiment 5: two-(2-OTBS xitix) the 6-ester (XXII) of synthesizing astaxanthin disuccinic acid ester

HPLC: post: 3.5 microns 4.6mm * 150mm of Waters Symmetry C18; Temperature: 25 ℃; Moving phase: (A=0.025%TFA is in water; B=0.025%TFA is in acetonitrile), 95%A/5%B (initial); Linear gradient to 100%B experience 5 minutes kept 10 minutes; Linear gradient to 95%B experience 2 minutes; Linear gradient to 95%A/5%B experience 3 minutes; Flow velocity: 1.0mL/min; Detector wavelength: 474nm.

Toward astaxanthin disuccinic acid ester (the XV) (20.00g in the 600mL methylene dichloride; 25.1mmol) the solution of stirring add 4-dimethylaminopyridine (DMAP) (6.13g; 50.2mmol), 2-O-t-butyldimethylsilyl (OTBS) xitix (XXVI) (21.86g; 75.3mmol) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI-HCl) (12.02g, 62.75mmol).With silica gel (1.0kg silica gel, eluent 0.5%HOAc/5%MeOH/EtOAc) reaction mixture being carried out flash chromatography after 14 hours handles.With the cut 10 concentrated garnet solids (6.47g, 19.2% productive rate are 58%AUC purity according to HPLC) that obtain.With silica gel (600g silica gel, eluent 0.25%HOAc/5%MeOH/EtOAc) crude product being carried out flash chromatography handles.Cut 6-10 vacuum concentration is obtained garnet solid (1.50g, 4.4% productive rate are 94.8%AUC purity according to HPLC). ¹H-NMR (CDCl ₃) δ 11.13 (2H, s), 6.78-6.28 (14H, m), 5.43 (2H, dd, J=12.2,7.1Hz); 5.34 (2H, s), 4.78 (2H, d, J=5.4Hz), 4.11-4.07 (6H, m); 2.69-2.65 (8H, m), 2.05-1.97 (22H, m), 1.81 (6H, s), 1.33 (6H; S), 0.92 (18H, s), 0.15 (6H, s), 0.14 (6H, s); HPLC 13.4 minutes [94.8% (AUC)]; Mass spectrum-ESI, m/z=1340.6 (M ^-).

Embodiment 6: two xitix 6-esters (XIX) of synthesizing astaxanthin disuccinic acid ester.

(100mg, stirred solution 0.075mmol) adds HFEt to 0 ℃ of following two-(2-OTBS xitix) 6-ester (XXII) toward the astaxanthin disuccinic acid ester in THF (5mL) ₃N (121 μ L, 0.745mmol).Reactant was stirred 1 hour down in 0 ℃, and temperature is to room temperature then.Reactant was stirred 2.5 hours, and the separating funnel that comprises 5mL IPAC and 5mL water through impouring then comes termination reaction.Remove water layer, and water (2 * 5mL) washing organic layers.Remove organic solvent through rotary evaporation, obtain a kind of garnet solid, it is purified and use; ¹H-NMR (CDCl ₃) δ 11.12 (2H, s), 8.40 (2H, s), 6.87-6.28 (14H, m), 5.43-5.32 (4H, m), 4.69 (s, 2H), 4.09 (s, 4H), 3.99 (s, 2H), 2.68-2.50 (m, 8H), 2.00-1.76 (22H, m), 1.36-1.19 (12H, m); HPLC 8.9 minutes [80.7% (AUC)]; Mass spectrum+ESI, m/z=1113.2 (M+H ⁺).

Embodiment 7: the sodium salt (XXIII) of two xitix 6-esters of synthesizing astaxanthin disuccinic acid ester.

Under the room temperature toward the solution of two xitix 6-esters (XIX) stirring (0.075mmol) of the bullion astaxanthin disuccinic acid ester in acetone (5mL) add triethyl orthoformate (62 μ L, 0.373mmol).With solution stirring 15 minutes, drip then the 2 ethyl hexanoic acid sodium in acetone solution (93 μ L, 0.019mmol, 0.20M).Through removing by filter the deposition of gained.Filtrating is cooled to 0 ℃, and (373 μ L, 0.075mmol handle 0.20M) with additional 2 ethyl hexanoic acid sodium in acetone.Reactant was stirred 5 minutes, through the solid collected by filtration material, wash then with acetone (5mL); And drying obtains a kind of garnet solid (27.8mg under high vacuum; 32.2% productive rate): HPLC 8.9 minutes [88.2% (AUC)], mass spectrum+APCI, m/z=1113.3 (M+3H-2Na ⁺).

Embodiment 8: the dicyclohexyl methyl esters (XXIV) of synthesizing astaxanthin disuccinic acid ester.

HPLC: post: Alltech Rocket; Platinum-C18;

3AM, 7 * 53mm; Temperature: 25 ℃; Moving phase: (A=0.025%TFA is in water; B=0.025%TFA is in acetonitrile), 70%A/30%B (initial); Kept 40 seconds; Linear gradient to 50%B experience 4 minutes and 20 seconds; Linear gradient to 100%B experience 1 minute and 30 seconds kept 4 minutes and 40 seconds; Linear gradient is to 70%A/30%B, 20 seconds; Flow velocity: 2.5mL/ branch; Detector wavelength: 474nm.

Room temperature and N ₂(100mg, 0.125mmol) and N, the solution of the stirring of dinethylformamide (6.0mL) adds cesium carbonate, and (90.0mg 0.275mmol), and covers with aluminium foil toward the astaxanthin disuccinic acid ester (XV) in the 25mL round-bottomed flask under the nitrogen atmosphere.Reactant was stirred 15 minutes, add then the brooethyl hexanaphthene (52.0 μ L, 0.375mmol).After 2 days, through adding 4mL saturated solution of sodium bicarbonate termination reaction, and dilute with the 50mL methylene dichloride.With the solution washing secondary of 25mL water, use anhydrous sodium sulfate drying then with dilution.Organic solution is filtered, and with the rotary evaporation method except that desolvating.With the thick resistates of flash chromatography (10-50%EtOAc/ heptane) purifying, obtain a kind of garnet solid (40.2mg, 32.5% productive rate): ¹H-NMR (CDCl ₃) δ 7.03-6.17 (14H, m), 5.54 (2H, dd, J=12.9,6.7Hz), 3.92 (4H, d, J=6.4Hz), 2.82-2.63 (8H, m), 2.08-1.92 (14H, m), 1.90 (6H, s), 1.75-1.62 (14H, m), 1.34-1.20 (22H, m); HPLC 8.9 minutes [83.9% (AUC)]; TLC (3: 7EtOAc/ heptane: R _f0.38); Mass spectrum+ESI, m/z=989.6 (M+H ⁺).

Embodiment 9: Synthetic 2-OTBS-5,6-isopropyledine xitix (XXV)

HPLC:Alltech Rocket, Platinum-C18,100A, 3 μ m, 7 * 53mm, PN50523; Temperature: 25 ℃; Moving phase: (A=0.025%TFA is in water; B=0.025%TFA is in acetonitrile), 90%A/10%B (initial); Linear gradient to 30%B experience 3 minutes; Linear gradient to 90%B experience 3 minutes kept 2 minutes; Linear gradient to 90%A/10%B experience 1 minute kept 1 minute then; Flow velocity: 2.5ml/ branch; Detector wavelength: 256nm.

Under the room temperature toward in 1.00L THF 5; 6-isopropyledine xitix (100.0g; The solution of stirring 463mmol) adds tert-butyldimethylsilyl chloride (TBSCl), and (76.7g 509mmol), added N then in 30 minutes; N-diisopropyl ethyl amine (DIPEA) (161mL, 925mmol).Reactant was stirred under room temperature 14 hours, then vacuum concentration.Mixture is dissolved in MTBE (MTBE) (1.00L), and in separating funnel, extracts with 1M salt of wormwood (1.00L).Use MTBE (1.00L) aqueous layer extracted more once, and with 2N HCl with the pH regulator of water layer to pH 6.With (1.00L) aqueous layer extracted secondary of isopropyl acetate (IPAC), and concentrate and obtain a kind of beige solid (150.4g, 98% productive rate): ¹H-NMR (DMSO d ₆) δ 11.3 (1H, s), 4.78 (1H, d, J=2.0Hz), 4.41-4.36 (1H, m), 4.11 (1H, dd, J=8.4,7.4Hz), 3.92 (1H, dd, J=8.4,6.0), 1.24 (3H, s), 1.23 (3H, s), 0.92 (9H, s), 0.14 (6H, s); HPLC 5.9 minutes [91.6% (AUC)]; Mass spectrum-ESI, m/z=329.2 (M-H ^-).

Embodiment 10: Synthetic 2-OTBS xitix (XXVI).

Under room temperature and the nitrogen atmosphere toward the 2-OTBS-5 in the 1.50L methylene dichloride, 6-isopropyledine xitix (XXV) (150.4g, the solution of stirring 455mmol) add dimercaptopropane (54.0mL, 546mmol).Solution is cooled to-45 ℃, drips BF with the speed that keeps temperature to be lower than-40 ℃ then ₃-OEt ₂(58.0mL, 455mmol).After 1 hour, react completely according to HPLC.Separating funnel through the cold reaction mixture impouring being contained 1.00L IPAC and 500mL saturated ammonium chloride solution and 500ml water comes termination reaction.Organic layer is concentrated into a kind of white solid.In order to remove dimercaptopropane,, add heptane (1.00L), and stirred 1 hour solid pulp 2 hours again in methylene dichloride (250mL).With the mixture vacuum concentration to the 500ml volume.Filtering mixture also, vacuum-drying obtains a kind of beige solid (112.0g, 85% productive rate): ¹H-NMR (DMSO d ₆) δ 11.0 (1H, s), 4.89 (2H, s), 4.78 (1H, d, J=1.2Hz), 3.82-3.80 (1H, m), 3.45-3.42 (2H, m), 0.923 (9H, s), 0.14 (6H, s); HPLC 4.9 minutes [92.0% (AUC)]; Mass spectrum-ESI, m/z=289.0 (M-H ^-).

Embodiment 11: the two-dimethyl-SULPHOSUCCINIC ACID ESTER (XXVII) of synthesizing astaxanthin.

HPLC:Waters Symmetry C18,3 μ m, 4.6 * 150mm, WAT200632, temperature: 25 ℃; Moving phase: (A=water; B=10%DCM/MeOH), 10%A/90%B (initial); Linear gradient to 100%B experience 9 minutes; Keep 100%B experience 11min, linear gradient to 10%A/90%B experience 1 minute; Flow velocity: 1.0ml/ branch; Detector wavelength: 474nm.

37 ℃ down toward the astaxanthin 2E in methylene dichloride (500mg, 0.84mmol) and Methylimidazole (0.50ml, 6.27mmol) mixture adding bromo dimethyl phosphate (2M, 5.04mL) (Ding, 2000).After 24 hours, incomplete according to the HPLC reaction, adding bromo dimethyl phosphate (2M, 5.04mL).After 48 hours, incomplete according to the HPLC reaction, and adding bromo dimethyl phosphate (2M, 5.04mL).After 72 hours, react completely according to HPLC.With methylene dichloride (20mL) diluting reaction thing, and water (20mL) termination reaction.Separate each layer and use 20mL dichloromethane extraction water layer once more.Merge organic layer, and under vacuum, concentrate and obtain 2.69g (＞100% productive rate). ¹H NMR (CDCl ₃) δ 6.58-6.14 (14H, m), 5.05-4.95 (2H, m), 3.91-3.60 (12H, m), 2.11-2.04 (4H, m), 2.04-1.92 (12H, m), 1.85 (6H, s), 1.26 (6H, s), 1.15 (6H, s) .HPLC:4.29 minute (86.7%AUC)).Moving phase: A=0.025%TFA is at H ₂Among the O; B=0.025%TFA, in acetonitrile, 10%A/90%B (initial); PDA detector 474nm.+ESI, m/z=813.62 (M+1).

Embodiment 12: the BocProOH ester (XXVIII) of synthesizing astaxanthin.

LC/MS analyzes: in Agilent 1100LC/MSD VL ESI system, carry out LC/MS and analyze, said system contains Zorbax Eclipse XDB-C18 Rapid Resolution4.6x75mm, 3.5 μ M, USUT002736; Temperature: 25 ℃; Moving phase: (%A=0.025%TFA is at H ₂Among the O; %B=0.025%TFA is in MeCN), 70%A/30%B (initial); Linear gradient experiences 5 minutes to 50%B, and linear gradient experiences 3 minutes to 98%B, and maintenance is 17 minutes under 98%B; Flow velocity: 1.0ml/ branch; Initial pressure: 108 crust; PDA detector 470nm, 373nm, 214nm.LRMS:+ pattern, ESI.

Toward the astaxanthin 2E (5.00g in methylene dichloride (500mL); 8.38mmol) and BocProOH (10.8g, (6.14g is 50.3mmol) with 1 for mixture adding 4-dimethylaminopyridine (DMAP) 50.3mmol); 3-di-isopropyl carbodiimide (DIC) (7.79mL, 50.3mmol).With mixture stirred overnight under room temperature and nitrogen atmosphere.After 16 hours, react completely according to TLC.Then mixture is concentrated into dried, and with the 100mL ether with thick resistates pulp, filter through the zeyssatite filter bed.With silica gel (Et ₂O) filtrating is carried out the flash chromatography processing and obtain title product, be a kind of garnet solid (8.56g,＞100% productive rate).LC:17.5 minute [23.1%AUC)]; Minute 18.2 [45.1% (AUC)]; 19.4 minute [22.0% (AUC)] .TLC (3: 2EtOAc/ hexane: R _f0.51; R _f0.55; Rf 0.59) .MS+ESI, m/z=1013.8 (M+Na ⁺).

Embodiment 13: the dihydrochloride (XXIX) of two proline esters of synthesizing astaxanthin.

(48.9mL, mixture 838mmol) are cooled to-78 ℃ with ether (130mL) and EtOH under nitrogen atmosphere.Past refrigerative mixture dripping acetyl chloride in 30 minutes (82.0mL, 838mmol).From cooling bath, shift out reactant, and be slowly to warm to room temperature.The flask contents impouring is contained the DiBocPro ester (XXVIII) of astaxanthin, and (8.31g is 8.38mmol) with the independent round-bottomed flask of stirring rod.Cover flask with aluminium foil, and with reactant stirred overnight under room temperature and nitrogen atmosphere.After 16 hours, react completely according to LC.Title compound precipitates and collects with filtration method, and (3 * 100mL) wash and dry (6.37g, 88.0% thick productive rate is 75.2% purity according to LC) .LC:8.00 minute [75.2% (AUC)] .MS+ESI m/z=791.7 (M+H with ether ⁺).

Embodiment 14: synthetic xenthophylls disuccinic acid ester (XXXII).

Under the room temperature toward the xenthophylls (XXX) in DCM (2mL) (0.010g, 0.018mmol) solution add DIPEA (0.063mL, 0.360mmol), succinyl oxide (0.036g, 0.360mmol) and DMAP (0.021g, 0.176mmol).

Reaction mixture was at room temperature stirred 48 hours, and, with salt solution/1M HCl (6mL/1mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.Use Na ₂SO ₄The dry organic layer that merges, and the concentrated xenthophylls disuccinic acid ester (XXXII) (93.09%) that obtains.The HPLC RT: 11.765 minutes, 93.09% (AUC); LRMS (ESI) m/z (relative intensity): 769 (M ⁺) (24), 651 (100), and do not have detectable xenthophylls.

Embodiment 15: the succinate of synthetic ZXN (XXXIII, XXXIV).

Under the room temperature toward the ZXN (XXXI) in DCM (2mL) (0.010g, 0.018mmol) solution add DIPEA (0.063ml, 0.360mmol), succinyl oxide (0.036g, 0.360mmol) and DMAP (0.021g, 0.176mmol).Reaction mixture was at room temperature stirred 48 hours, and, with salt solution/1M HCl (6mL/1mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.Use Na ₂SO ₄The dry organic layer that merges, and concentrated ZXN one succinate (XXXIII) (2.86%) that obtains.The HPLC RT: 12.207 minutes, 2.86% (AUC); LRMS (ESI) m/z (relative intensity): 669 (M ⁺+ H) (53), 668 (M ⁺) (100), ZXN disuccinic acid ester (XXXIV) (97.14%) HPLC RT: 11.788 minutes, 67.42% (AUC); LRMS (ESI) m/z (relative intensity): 792 (M ⁺+ Na) (42), 769 (M ⁺) (73), 651 (100); The HPLC RT: 13.587 minutes, 11.19% (AUC); LRMS (ESI) m/z (relative intensity): 792 (M ⁺+ Na) (36), 769 (M ⁺) (38), 663 (100); The HPLC RT: 13.894 minutes, 18.53% (AUC); LRMS (ESI) m/z (relative intensity): 769 (M ⁺) (62), 663 (77), 651 (100), and do not have detectable ZXN

Embodiment 16: and the aconitate of synthesizing astaxanthin (XXXV, XXXVI).

Under the room temperature toward at DCM/DMF (" N; the astaxanthin 2E of dinethylformamide ") in (4ml/2mL) (0.100g, solution 0.168mmol) add DIPEA (0.878mL, 5.04mmol), cis-aconitic anhydride (0.2622g; 1.68mmol) and DMAP (0.4105g, 3.36mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain equisetic acid one ester (XXXV) (13.25%) HPLC RT: 10.485 minutes, 4.95% (AUC); LRMS (ESI) m/z (relative intensity): 777 (M ⁺+ Na+2H) (57), 623 (100); The HPLC RT: 10.722 minutes, 8.30% (AUC); LRMS (ESI) m/z (relative intensity): 777 (M ⁺+ Na+2H) (6), 709 (100), equisetic acid diester (XXXVI) (27.67%) HPLC RT: 9.478 minutes, 15.44% (AUC); LRMS (ESI) m/z (relative intensity): 933 (M ⁺+ Na+2H) (10), 831 (100); The HPLC RT: 9.730 minutes, 12.23% (AUC); LRMS (ESI) m/z (relative intensity): 913 (M ⁺+ 4H) (4), 843 (100), and astaxanthin (44.40%).

Embodiment 17: the citrate of synthesizing astaxanthin (XXXVII, XXXVIII).

Under the room temperature toward the Hydrocerol A in DCM (8mL) (0.5149g, 2.86mmol) suspension-s add DIPEA (1.167mL, 0.6.70mmol), DIC (0.525mL, 3.35mmol), DMAP (0.4094g, 3.35mmol) and astaxanthin (0.200g, 0.335mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1MHCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain Hydrocerol A one ester (XXXVII) (26.56%) HPLC RT: 9.786 minutes, 17.35% (AUC); LRMS (ESI) m/z (relative intensity): 773 (M ⁺+ 3H) (14), 771 (M ⁺+ H) (100); The HPLC RT: 9.989 minutes, 9.21% (AUC); LRMS (ESI) m/z (relative intensity): 773 (M ⁺+ 3H) (50), 771 (M ⁺+ H) (100), Hydrocerol A diester (XXXVIII) (7.81%) HPLC RT: 8.492 minutes, 3.11% (AUC); LRMS (ESI) m/z (relative intensity): 968 (M ⁺+ Na) (75), 967 (100), 946 (M ⁺+ H) (37); The HPLC RT: 8.708 minutes, 2.43% (AUC); LRMS (ESI) m/z (relative intensity): 968 (M ⁺+ Na) (95), 946 (M ⁺+ H) (100); The HPLC RT: 8.952 minutes, 2.27% (AUC); LRMS (ESI) m/z (relative intensity): 946 (M ⁺+ H) (19), 500 (100), and astaxanthin (21.26%).

Embodiment 18: dimethylaminobutyricacid acid one ester (XXXIX) of synthesizing astaxanthin.

Under the room temperature toward 4-(the dimethylamino)-butyrates hydrochlorate in DCM/DMF (3mL/3mL) (0.2816g, 1.68mmol) suspension-s add DIPEA (0.878mL, 5.04mmol), HOBT (" I-hydroxybenzotriazole ")-H ₂O (0.3094g, 2.02mmol), DMAP (0.4105g, 3.36mmol) and astaxanthin (0.100g, 0.168mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1M HCl (20ml/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain 4-(dimethylamino) butyric acid one ester (XXXIX) (24.50%) HPLC RT: 9.476 minutes, 20.32% (AUC); LRMS (ESI) m/z (relative intensity): 732 (M ⁺+ Na) (13), 729 (100); The HPLC RT: 9.725 minutes, 4.18% (AUC); LRMS (ESI) m/z (relative intensity): 732 (M ⁺+ Na) (50), 729 (100), and astaxanthin (61.21%).

Embodiment 19: gsh one ester (L) of synthesizing astaxanthin.

Under the room temperature toward the reduced glutathione in DCM/DMF (3mL/3mL) (0.5163g, 1.68mmol) suspension-s add DIPEA (0.878mL, 5.04mmol), HOBT-H ₂O (0.3094g, 2.02mmol), DMAP (0.4105g, 3.36mmol), DIC (0.316mL, 2.02mmol) with astaxanthin 2E (0.100g, 0.168mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1MHCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain gsh one ester (L) (23.61%) HPLC RT: 9.488 minutes, 16.64% (AUC); LRMS (ESI) m/z (relative intensity): 886 (M ⁺) (13), 810 (54), 766 (100); The HPLC RT: 9.740 minutes, 3.57% (AUC); LRMS (ESI) m/z (relative intensity): 886 (M ⁺) (24), 590 (78), 546 (100); The HPLC RT: 9.997 minutes, 3.40% (AUC); LRMS (ESI) m/z (relative intensity): 886 (M ⁺) (25), 869 (85), 507 (100), and astaxanthin (68.17%).

Embodiment 20: diester tartaric acid used (LI) of synthesizing astaxanthin.

Under the room temperature toward (the L)-tartrate in DCM/DMF (5mL/5mL) (0.4022g, 2.68mmol) suspension-s add DIPEA (1.167mL, 0.6.70mmol), HOBT-H ₂O (0.5131g, 3.35mmol), DMAP (0.4094g, 3.35mmol) with astaxanthin 2E (0.200g, 0.335mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain diester tartaric acid used (LI) (18.44%) HPLC RT: 9.484 minutes, 14.33% (AUC); LRMS (ESI) m/z (relative intensity): 884 (M ⁺+ Na+H) (100), 815 (72), 614 (72); The HPLC RT: 9.732 minutes, 4.11% (AUC); LRMS (ESI) m/z (relative intensity): 883 (M ⁺+ Na) (100), 539 (72), and astaxanthin (67.11%).

Embodiment 21: sorbyl alcohol one ester (LII) of synthesizing astaxanthin disuccinic acid ester.

Under the room temperature toward the astaxanthin disuccinic acid ester (XV) in DMF (10mL) (0.200g, 0.251mmol) solution add DIPEA (1.312mL, 7.53mmol), HOBT-H ₂O (0.4610g, 3.01mmol), DMAP (0.6133g, 5.02mmol) with (D)-sorbyl alcohol (0.4572g, 2.51mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain sorbyl alcohol one ester (LII) (3.52%) HPLC RT: 9.172 minutes, 3.52% (AUC); LRMS (ESI) m/z (relative intensity): 984 (M ⁺+ Na) (28), 503 (100) and astaxanthin disuccinic acid ester (91.15%).

Embodiment 22: the sorbyl alcohol diester (LIII) of synthesizing astaxanthin disuccinic acid ester.

Under the room temperature toward the astaxanthin disuccinic acid ester (XV) in DCM/DMF (3mL/3mL) (0.100g, 0.125mmol) solution add DIPEA (0.656mL, 3.76mmol), HOBT-H ₂O (0.2313g, 1.51mmol), DMAP (0.3067g, 2.51mmol), DIC (0.236mL, 1.51mmol) with (D)-sorbyl alcohol (0.2286g, 1.25mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain sorbyl alcohol diester (LIII) (44.59%) HPLC RT: 8.178 minutes, 11.58% (AUC); LRMS (ESI) m/z (relative intensity): 1148 (M ⁺+ Na) (40), 545 (100); The HPLC RT: 8.298 minutes, 33.01% (AUC); LRMS (ESI) m/z (relative intensity): 1148 (M ⁺+ Na) (20), 545 (100), do not have detectable astaxanthin disuccinic acid ester.

Embodiment 23: the morpholine carbamate of synthesizing astaxanthin (LIV, LV).

Under the room temperature toward the astaxanthin 2E in DCM/DMF (3mL/3mL) (0.100g, 0.168mmol) solution add DIPEA (0.878mL, 5.04mmol), DMAP (0.4105g, 3.36mmol) with 4-morpholine carbonyl chloride (0.196mL, 1.68mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain 4-morpholine one carbamate (LIV) (33.17%) HPLC RT: 11.853 minutes, 29.01% (AUC); LRMS (ESI) m/z (relative intensity): 710 (M ⁺) (100); The HPLC RT: 13.142 minutes, 1.37% (AUC); LRMS (ESI) m/z (relative intensity): 710 (M ⁺) (100); The HPLC RT: 13.383 minutes, 2.79% (AUC); LRMS (ESI) m/z (relative intensity): 710 (M ⁺) (100), 4-morpholine diurethanes (LV) (33.42%) HPLC RT: 12.049 minutes, 29.71% (AUC); LRMS (ESI) m/z (relative intensity): 824 (M ⁺+ H) (54), 823 (M ⁺) (100); The HPLC RT: 13.761 minutes, 1.29% (AUC); LRMS (ESI) m/z (relative intensity): 823 (M ⁺) (100), 692 (75); The HPLC RT: 14.045 minutes, 2.42% (AUC); LRMS (ESI) m/z (relative intensity): 823 (M ⁺) (100), 692 (8), and astaxanthin (22.10%).

Embodiment 24: N.F,USP MANNITOL one carbonic ether (LVII) of synthesizing astaxanthin.

0 ℃ down toward the astaxanthin 2E in DCM (4mL) (0.100g, solution 0.168mmol) add DIPEA (0.585mL, 3.36mmol) and 1,2,2,2-tetrachloro ethyl chloroformate (0.103mL, 0.672mmol).Reaction mixture was stirred 2 hours down in 0 ℃, at room temperature stirred 1.5 hours, this moment with (D)-N.F,USP MANNITOL (0.3060g, 1.68mmol), (0.2052g 1.68mmol) is added to reactant for DMF (3mL) and DMAP.Reaction mixture was at room temperature stirred 24 hours, and, with salt solution (20mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain N.F,USP MANNITOL one carbonic ether (LVII) (10.19%) HPLC RT: 9.474 minutes, 10.19% (AUC); LRMS (ESI) m/z (relative intensity): 827 (M ⁺+ Na) (50), 804 (M ⁺) (25), 725 (58), 613 (100), and astaxanthin (53.73%).

Embodiment 25: (dimethylamino) butyric acid diester (LVIII) of synthesizing astaxanthin.

Under the room temperature toward 4-(dimethylamino) the butyrates hydrochlorate in DCM/DMF (3mL/3mL) (0.2816g, 1.68mmol) suspension-s add DIPEA (0.878ML, 5.04mmol), DMAP (0.4105g, 3.36mmol), HOBT-H ₂O (0.3094g, 2.02mmol), DIC (0.316mL, 2.02mmol) with astaxanthin 2E (0.100g, 0.168mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1MHCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain (dimethylamino) butyric acid diester (LVIII) (77.70%) HPLC RT: 7.850 minutes, 56.86% (AUC); LRMS (ESI) m/z (relative intensity): 824 (M ⁺+ H) (64), 823 (M ⁺) (100); The HPLC RT: 8.443 minutes, 3.87% (AUC); LRMS (ESI) m/z (relative intensity): 823 (M ⁺) (5), 641 (20), 520 (100); The HPLC RT: 9.021 minutes, 16.97% (AUC); LRMS (ESI) m/z (relative intensity): 824 (M ⁺+ H) (58), 823 (M ⁺) (100), and do not have detectable astaxanthin.

Embodiment 26: benzyl one ether (LIX) of synthesizing astaxanthin.

0 ℃ down toward the astaxanthin 2E in DCM/DMF (3mL/3mL) (0.100g, 0.168mmol) and bromotoluene (0.400mL, solution adding KHMDS (" two (trimethyl silyl) acid amides potassium ") (6.72mL 3.36mmol); 0.5M, in toluene, 3.36mmol).Reaction mixture was stirred 1 hour down in 0 ℃, and temperature is to room temperature then.Mixture was stirred under room temperature 24 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain benzyl one ether (LIX) (15.06%) HPLC RT: 12.705 minutes, 15.06% (AUC); LRMS (ESI) m/z (relative intensity): 686 (M ⁺) (93), 597 (100), and astaxanthin (67.96%).

Embodiment 27: N.F,USP MANNITOL one ether (LX) of synthesizing astaxanthin.

(0.200g, solution 0.335mmol) adds 48%HBr (10mL) and H toward the astaxanthin 2E in DCM (15mL) under the room temperature ₂O (30mL).Use the DCM aqueous layer extracted, use Na ₂SO ₄The dry organic layer that merges, and concentrate the bromide verivate that obtains astaxanthin, be a kind of garnet oil.Under the room temperature toward the solution of the thick bromide in DCM/DMF (6mL/6mL) add DIPEA (1.58ML, 9.09mmol), DMAP (0.3702g, 3.03mmol) with (D)-N.F,USP MANNITOL (0.5520g, 3.03mmol).Reaction mixture was at room temperature stirred 24 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain N.F,USP MANNITOL one ether (LX) (4.40%) HPLC RT: 9.479 minutes, 4.40% (AUC); LRMS (ESI) m/z (relative intensity): 783 (M ⁺+ Na) (64), 710 (66), 653 (100), and astaxanthin (79.80%).

Embodiment 28: three (hydroxymethyl) the aminomethane monoamide (LXI) of synthesizing astaxanthin disuccinic acid ester.

Under the room temperature toward the astaxanthin disuccinic acid ester (XV) in DCM/DMF (3mL/3mL) (0.100g, 0.125mmol) solution add DIPEA (0.653mL, 3.75mmol), DMAP (0.3054g, 2.50mmol), HOBT-H ₂O (0.2297g, 1.50mmol) with three (hydroxymethyl) aminomethane (0.1514g, 1.25mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain three (hydroxymethyl) aminomethane monoamide (LXI) (4.40%) HPLC RT: 9.521 minutes, 3.50% (AUC); LRMS (ESI) m/z (relative intensity): 923 (M ⁺+ Na) (36), 900 (M ⁺) (80), 560 (100); The HPLC RT: 9.693 minutes, 0.90% (AUC); LRMS (ESI) m/z (relative intensity): 923 (M ⁺+ Na) (11), 813 (33), 500 (100) and astaxanthin disuccinic acid ester (84.34%).

Embodiment 29: three (hydroxymethyl) the aminomethane diamide (LXII) of synthesizing astaxanthin disuccinic acid ester.

Under the room temperature toward the astaxanthin disuccinic acid ester (XV) in DCM/DMF (3mL/3mL) (0.100g, 0.125mmol) solution add DIPEA (0.653mL, 3.75mmol), DMAP (0.3054g, 2.50mmol), HOBT-H ₂O (0.2297g, 1.50mmol), DIC (0.235ML, 1.50mmol) with three (hydroxymethyl) aminomethane (0.1514g, 1.25mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain three (hydroxymethyl) aminomethane diamide (LXII) (66.51%) HPLC RT: 8.086 minutes, 19.34% (AUC); LRMS (ESI) m/z (relative intensity): 1026 (M ⁺+ Na) (22), 1004 (M ⁺+ H) (84), 1003 (M ⁺) (100), 502 (83); The HPLC RT: 8.715 minutes, 47.17% (AUC); LRMS (ESI) m/z (relative intensity): 1004 (M ⁺+ H) (71), 1003 (M ⁺) (100), 986 (62) and astaxanthin disuccinic acid ester (18.61%).

Embodiment 30: adenosine one ester (LXIII) of synthesizing astaxanthin disuccinic acid ester.

Under the room temperature toward the astaxanthin disuccinic acid ester (XV) in DCM/DMF (3mL/3mL) (0.100g, 0.125mmol) solution add DIPEA (0.653mL, 3.75mmol), DMAP (0.3054g, 2.50mmol), HOBT-H ₂O (0.1914g, 1.25mmol) with (-)-adenosine (0.3341g, 1.25mmol).Reaction mixture was at room temperature stirred 48 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain adenosine one ester (LXIII) (21.13%) HPLC RT: 9.005 minutes, 2.43% (AUC); LRMS (ESI) m/z (relative intensity): 1047 (M ⁺+ H) (36), 1046 (M ⁺) (57), 524 (100); The HPLC RT: 9.178 minutes, 10.92% (AUC); LRMS (ESI) m/z (relative intensity): 1047 (M ⁺+ H) (80), 1046 (M ⁺) (100), 829 (56), 524 (94); The HPLC RT: 9.930 minutes, 7.78% (AUC); LRMS (ESI) (relative intensity): 1046 (M ⁺) (100), 524 (34) and astaxanthin disuccinic acid ester (58.54%).

Embodiment 31: the SANMALT-S diester (LXIV) of synthesizing astaxanthin disuccinic acid ester.

Under the room temperature toward the astaxanthin disuccinic acid ester (XV) in DCM/DMF (3mL/3mL) (0.100g, 0.125mmol) solution add DIPEA (0.653mL, 3.75mmol), DMAP (0.3054g, 2.50mmol), HOBT-H ₂O (0.2297g, 1.50mmol), DIC (0.235mL, 1.50mmol) with (D)-SANMALT-S-H ₂O (0.4504g, 1.25mmol).Reaction mixture was at room temperature stirred 36 hours, and, with salt solution/1M HCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain SANMALT-S diester (LXIV) (25.22%) HPLC RT: 7.411 minutes, 12.53% (AUC); LRMS (ESI) m/z (relative intensity): 1468 (M ⁺+ Na) (18), 1067 (16), 827 (100); The HPLC RT: 7.506 minutes, 12.69% (AUC); LRMS (ESI) m/z (relative intensity): 1468 (M ⁺+ Na) (52), 827 (76), 745 (100) and astaxanthin disuccinic acid ester (22.58%).

Embodiment 32: synthesizing astaxanthin disuccinic acid ester from the black false hellebore alcohol ester (LXV, LXVI).

Under the room temperature toward the astaxanthin disuccinic acid ester (XV) in DCM/DMF (3mL/3mL) (0.100g, 0.125mmol) solution add DIPEA (0.653mL, 3.75mmol), DMAP (0.3054g, 2.50mmol), HOBT-H ₂O (0.2297g, 1.50mmol), DIC (0.235mL, 1.50mmol) and trans-resveratrol (0.2853g, 1.25mmol).Reaction mixture was at room temperature stirred 24 hours, and, with salt solution/1MHCl (20mL/3mL) termination reaction then with DCM extract with DCM diluting reaction thing this moment.The organic layer that merges concentrated obtain trans-resveratrol one ester (LXV) (1.12%) HPLC RT: 10.039 minutes, 1.12% (AUC); LRMS (ESI) m/z (relative intensity): 1009 (M ⁺+ 2H) (18), 1007 (M ⁺) (21), 637 (100), trans-resveratrol diester (LXVI) (60.72%) HPLC RT: 10.324 minutes, 15.68% (AUC); LRMS (ESI) m/z (relative intensity): 1217 (M ⁺) (28), 1007 (100), 609 (69), 504 (85); The HPLC RT: 10.487 minutes, 29.26% (AUC); LRMS (ESI) m/z (relative intensity): 1218 (M ⁺+ H) (80), 1217 (M ⁺) (100), 609 (60); The HPLC RT: 10.666 minutes, 15.78% (AUC); LRMS (ESI) m/z (relative intensity): 1218 (M ⁺+ H) (84), 1217 (M ⁺) (100), 609 (71), and do not have detectable astaxanthin disuccinic acid ester.

Accurately measure the water-soluble of disodium disuccinic acid ester astaxanthin derivatives (XVI):

With the sample of 30mg (disodium disuccinic acid ester astaxanthin derivatives altogether; Be the steric isomer 3S of 1: 2: 1 ratio; 3 ' S, meso and 3R, the alltrans mixture of 3 ' R) is added to (0.2 μ M

) de-ionized (DI) water of the 2mL sterile filtration in 15mL glass centrifuge tube.Pipe is wrapped in the aluminium foil, and with mixture vibration 2 hours under 3500rpm centrifugal 10 minutes then.The aqueous solution is filtered through 0.45 micron PVDF throw-away-type strainer.Then the filtrating of 1ml volume is diluted with DI water aptly, and under 480nM, measure the concentration of solution, adopt four point calibration curves by the fresh sample preparation.After considering dilution, the concentration of the saturated solution of disodium disuccinic acid ester astaxanthin derivatives is 8.64mg/mL.

Suppress and/or improve the experimental data of disease:

Relatively radical-positively charged ion forms ability: non-esterified free astaxanthin and diacid disuccinic acid ester astaxanthin, use flash photolysis:

Figure 27 and Figure 28 are described in acquisition about the triplet state of non-esterified free astaxanthin and diacid disuccinic acid ester astaxanthin derivatives and the flash photolysis spectroscopic analysis result afterwards of carrotenoid radical cation state formation.The formation of carrotenoid radical cation is the measurement index as the potential source biomolecule physical behavior of the novel derivative of inhibitor.If the anti-oxidant behavior of the free astaxanthin that the verivate maintenance is non-esterified; Then all previous (being the document precedent) that confirm are used reasonably to imagine about the treatment of astaxanthin and are used for novel derivative, comprise singlet oxygen quencher at least, lipid peroxidation splitting of chain and/or directly free radical scavenging.

Direct irradiation carrotenoid (car) do not cause forming the carrotenoid triplet state ( ³Cars); Need a kind of photosensitizers.In this experiment, nitronaftalin (NN) is used as photosensitizers.After the irradiation, the photosensitizers of being excited (NN*) formation photosensitizers triplet state ( ³NN).When ³When NN meets with carrotenoid, take place with ³The energy of NN and electron-transfer reaction.Bring the metastable of detection gained through the characteristic absorption spectrum ³Car and carrotenoid radical cation (car ^.+).Non-polar solvent (for example hexane) helps ³The formation of car helps car and have more polar solvent (alcohol, water) ^.+Formation.Radical anion (the NN of photosensitizers ^.-) do not see usually owing to hang down uptake factor.

A. the spectrum of astaxanthin disuccinic acid (astaCOOH)

The transient absorption spectrum of astaCOOH in acetonitrile (MeCN), sensitizing agent NN.

Negative peak proof NN in the spectrum and the ground state consumption of astaCOOH.The posivtive spike at 550nm place shows formation astaCOOH triplet state; The posivtive spike at 850nm place shows formation astaCOOH radical cation. ³The car decay phase is when rapid.Behind the 15 μ s, half the ³Car disappears, and behind the 50 μ s, does not have ³The car residue.Car ^.+Be stable in the scope at this moment.

B. the spectrum of reference compound [non-esterified free astaxanthin (asta)].

The transient absorption spectrum of asta in acetonitrile (MeCN), sensitizing agent NN.

The spectrum of asta is approaching identical with the spectrum of astaCOOH.Behind the 50 μ s, ³Car disappears.At this moment in the scope, car ^.+Be stable.Negative, positive peak about in the absorption spectrum of astaCOOH and asta can be overlapping.

Flash photolysis result briefly discusses:

As if the difference that between flash photolysis experimental session diacid disuccinic acid ester astaxanthin derivatives (astaCOOH) and non-esterified free astaxanthin (asta), exists very little.The performance similar asta of AstaCOOH in the flash photolysis experiment.Therefore, with succsinic acid the free astaxanthin esterification is not changed optical physics performance and radical cation life-span.Two kinds of compounds all are that light is stable at the flash photolysis experimental session.Disuccinic acid ester astaxanthin derivatives keeps effective antioxygenic potential of astaxanthin, and under the esterification state, has activity.Therefore can regard it as a kind of " soft " medicine (as activity being arranged) through the entity of modifying, rather than prodrug, be used for treatment and use the performance (being water and fat free radical scavenging mutually) of giving the valuable two-phase free radical scavenging activity of this verivate.

Induce and connect the protein 43 protein expression:

People such as Rogers (1990) describe in detail about cell cultures, the method that Western blotting, quantitative photodensitometry and total protein are estimated, and Bertram (1999) has proposed modification.In brief, in the 4mL cell culture system with following treated MEC CH3/10T ^1/2Cell, substratum comprise 2% calf serum:

[1.TTNPB p-(E)-2-(5,6,7,8-tetrahydrochysene-5,5,8,8-tetramethyl--2-naphthyl) propenylbenzene formic acid] 10 ^-8M is in acetone [about connecting the positive control (4 μ l are in 4mL) that protein 43 raises]

2. disodium salt disuccinic acid ester astaxanthin derivatives/H ₂O, concentration 10 ^-5M (40 μ L are in 4mL)

3. disodium salt disuccinic acid ester astaxanthin derivatives/H ₂O, concentration 10 ^-6M (4 μ L are in 4mL)

4. disodium salt disuccinic acid ester astaxanthin derivatives/H ₂O, concentration 10 ^-7M (dilution in 1: 10,4 μ L are in 4mL)

5. disodium salt disuccinic acid ester astaxanthin derivatives H ₂O/ ethanol [EtOH] prescription, concentration 10 ^-5M (40 μ L are in 4mL)

6. disodium salt disuccinic acid ester astaxanthin derivatives H ₂The O/EtOH prescription, concentration 10 ^-6M (4 μ L are in 4mL)

7. aseptic H ₂O contrasts (40 μ L are in 4mL)

8. aseptic H ₂O/EtOH contrasts (20 μ L EtOH, 20 μ L H ₂O is in 4mL)

9. substratum contrast (4mL)

Collecting cell after cultivating 96 hours with test-compound and contrast solution.The color of all culture medium solutions is identical, but with 10 ^-5After two kinds of processing carrying out of disodium salt disuccinic acid ester astaxanthin derivatives of dilution color subjective become orange red.Find that with light microscope the cell that TTNPB handles is striated, prove myocyte's differentiation, this is the expected results in this cell culture system.After results and sedimentation cell, comprise two kind 10 ^-5The pipe of disodium salt disuccinic acid ester astaxanthin derivatives solution is a shiny red; Two kind 10 ^-6Dilution tube is a pink.About what other coloured carrotenoid confirmed, this was the subjective evidence that cell is taken in test-compound as before.

Dissolved cell makes the various albumen of 50 μ g on 10% polyacrylamide gel, carry out electrophoresis then.Then gel is transferred to the nitrocellulose filter.Measure total protein (Figure 29 with Coomassie blue stain; Swimming lane 6,7 and 9 fogs after gel shifts, and is not included in [Figure 31] in the quantitative comparison).Carry out the Western blotting with the anti-protein 43 antibody that connects, on the Biorad imager, carry out HRP chemoluminescence (Figure 30) then.Original gel is peeled off (stripped) once, and repeat Western trace twice, observe then.The result is contrasted (EtOH/H with respect to the swimming lane that shows the proteic background constitutive expression of connection protein 43 under the collating condition (no test-compound) 8 ₂O) carry out normalization method.By positive control and test-compound cause relative to be connected protein 43 inductive result shown in figure 31.

Cx43 result briefly discusses.

All are tried disodium salt disuccinic acid ester astaxanthin derivatives preparation and all induce and connect protein 43 and express to exceed in water and ethanol/water contrast and form expression levels (Figure 31).There is not true treatment effect (null hypothesis contrast μ ₁=handle average μ ₂) situation under detect under 5 kinds of independent respectively test conditionss that to induce the possibility that connects the protein 43 protein expression be 1/2 ⁵Or p=0.03.The disodium salt disuccinic acid ester astaxanthin derivatives of in water, preparing is all induced under each test conditions and is connected the protein 43 protein expression (from 10 ^-5To 10 ^-7M).The reduction of the minimum disodium salt disuccinic acid ester astaxanthin derivatives of test/water combination shows the dose-dependently of inductive reaction.

Final alcohol concn in being used in substratum is to induce rising relatively under the 0.5% single test conditions estimated.This discovery highly points out the bioavailability of this prescription to increase, because known ethanol reduces the gathering of disodium salt disuccinic acid ester astaxanthin derivatives in the aqueous solution.Concentration is greater than 10 in water ^-7With concentration in ethanol/water combination be 10 ^-5The solution of disodium salt disuccinic acid ester astaxanthin derivatives as if having than positive TTNPB contrast higher induce level.TTNPB is a kind of retinoid of efficient, it at 96 hours time points with 10 ^-8M induces effectively and connects the protein 43 expression.

Disodium salt disuccinic acid ester astaxanthin derivatives inducing to iuntercellular gap junction communication (GJC) in the l cell:

Carry out a series of experiments and induce the ability of the gap junction communication (GJC) in the immortal cell line of mouse fibroblast cell to estimate disodium salt disuccinic acid ester astaxanthin derivatives.Study:

(1), increases measurement cell/cell communication through the dye transfer between the fused cell in the monolayer culture thing at functional level;

(2), induce the ability that connects protein 43 (Cx43) protein expression to measure through these compounds at molecular level.Cx43 is the structural unit that allows intercellular channel in these inoblasts of GJC;

(3) at cell levels, shown in the quantity of the Cx43 immunoreactivity patch in the plasma membrane zone that disodium salt disuccinic acid ester astaxanthin derivatives increases with flanking cell directly contacts and big or small ability.

(1) communication analysis.Experimentize and strengthen MEC C3H/10T to estimate disodium salt disuccinic acid ester astaxanthin derivatives [as alltrans (full E) steric isomer, S, S ', meso and R, the statistics mixture of 1: 2: 1 ratio of R '] ¹/ ₂Gap between the cell connects the ability of cell-cell communication (GJC).The ability height correlation (Zhang, 1992) that this ability is previous and carrotenoid inhibition carcinogens inductive tumour transforms.And the connection communication between the myocardial cell of Cx43 mediation is responsible for keeping synchronous and the transfer (Peters, 1995) that prevents ARR signal.

Basic as aforementioned (Zhang, 1994) through (MO) microinjection to individual fused cell is analyzed the connection permeability for Sigma, St.Louis with optical dye fluorescent yellow CH.In brief, with following reagent the fusion culture of C3H/10T1/2 cell was handled 4 days: (1) is dissolved in 1: 2 ethanol/water (EtOH/H ₂O) the disodium salt disuccinic acid ester astaxanthin derivatives (1 * 10 of prescription ^-5M); (2) be dissolved in a kind of synthetic retinoid-TTNPB (1 * 10 of THF ^-8M), as positive control; Or (3) 1: 2EtOH/H ₂The cell that O handles is as negative control.Under differing camera lens, identify the single cell in each petridish, and (Eppendorf, Hamburg Germany) carry out vasopressing injection to use the microinjection pin that loads as the optical dye fluorescent yellow of 10% solution.Control pin through remote control micromanipulator, and cell and microscope are placed on the pneumatic anti-vibration platform.Successfully inject fluorescent yellow through proving with the brief illumination of the UV light that causes fluorescent yellow yellow fluorescence.This dyestuff is enough little, connects to pass through the gap, and charged, thus can only get into and inject the adjacent cell of cell, if they are in the connection communication.After allowing to connect 2 minutes that shift, under the UV illumination, take digital image.Confirm through using no inclined to one side density threshold method and SigmaScan software program (Jandel Scientific) to carry out digital image analysis after the number of the fluorocyte adjacent with the injection cell.The number of this communication cell is used the index that connects communication, like aforementioned (Hossain, 1993).

The result of this analysis shows, is dissolved in 1: 2EtOH/H ₂The disodium salt disuccinic acid ester astaxanthin derivatives (1 * 10 of O prescription ^-5M) effectively increase the degree that connects communication, surpass 1: 2EtOH/H ₂Being seen degree in the control group that O handles.In the cell that 22 microinjections are handled, there are 15 (56%) to connect and functional coupling through the gap, control cells only has 3/11sts (27%) by contrast.These difference are (p＜0.04 discrepant on the statistics; Paired Student ' s t check).Representative Photomicrograph is shown in figure 14:

The A group: using concentration is 1 * 10 ^-5M 1: 2EtOH/H ₂The statistics mixture process of the steric isomer of the disodium salt disuccinic acid ester astaxanthin among the O;

C group: 1: 2EtOH/H ₂O solvent negative control;

The E group: TTNPB, concentration is 1 * 10 ^-8M, in THF as solvent, positive control; With

B, D, F group: difference numerical analysis Photomicrograph A, C, E, being presented at the above pixel of threshold value is set is fluorescent yellow fluorescence male.Because nucleus has maximum volume, they accumulate maximum fluorescent yellows, and the performance maximum fluorescence.

(2) molecular studies.The disodium salt disuccinic acid ester astaxanthin derivatives (S of the mixture of the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives and the enantiomeric forms of purifying; S ', meso and R; R ' form; According to HPLC purity＞90%) increase the proteic expression of Cx43 in the mouse fibroblast cell, it is by basically like immunity (Western) the blotting evaluation of said (Zhang, 1992 and 1994).In brief, at additional 5% foetal calf serum (AtlantaBiologicals, Atlanta; GA) and 25 μ g/mL GT (Sigma; St.Louis, MO) contain Earle salt (Atlanta Biologicals, Atlanta; GA) cultivate MEC C3H/10T1/2 cell in the Eagle minimum medium, and under 37 ℃ at 5%CO ₂The middle cultivation.Inoculation back is the 7th day in 100 millimeters (mm) plates, with disodium salt disuccinic acid ester astaxanthin derivatives with fused cell processing 4 days, then like said ground harvested cell and to analyze Cx43 protein induced.(Pierce Chemical Co., Rockford IL) measure protein content with the analysis of protein test kit according to the indication of manufacturers.Through the Western blotting; Use NuPage western trace test kit and instrument (Invitrogen; Carlsbad CA) analyzes and to comprise the proteic cell lysates of 100 μ g, and with to the rabbit polyclonal antibody (Zymed corresponding to the synthetic polypeptide generation in the C-terminal territory of mouse, people and rat Cx43; San Francisco CA) detects Cx43 albumen.Use is combined with the anti-rabbit two of HRP, and anti-(Pierce Chemical Co., Rockford IL) observe Cx43 immunoreactivity band through chemoluminescence.Obtain digital picture with refrigerative CCD camera, carry out then quantitative spectrodensitometry (Bio-Rad, Richmond, CA).Through identical with the albumen carrying capacity of digital image analysis proof swimming lane with the dyeing of Coomassie blue protein dyestuff.

This experiment in, with disodium salt disuccinic acid ester astaxanthin derivatives with 1: 2 ethanol/H ₂O prescription, concentration are 1 * 10 ^-5M is added to cell culture.The statistics mixture of steric isomer and the enantiomeric forms of purifying show with separately with 1: 2 ethanol/H ₂The expression that the cell culture that O handles is compared Cx43 increases (Figure 15 A and Figure 15 B).Statistics mixture process with the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives is tried to cause in the verivate that at all the highest Cx43 induces level.These are induced level and are included as retinoid tetrahydrochysene tetramethyl-naphthyl propenylbenzene formic acid (the TTNPB) (Hoffman-LaRoche of positive control than using; Nutley; NJ) and retinoic acid ester (Sigma, St.Louis, MO) the being seen little several times of level of inducing; This relative effectivenes difference is consistent with previous research.

(3) cell research.The statistics mixture of the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives increases the assembling of Cx43 in cell/cells contacting zone in the mouse 10T1/2 cell of handling, consistent with the formation that functional clearance connects.

In this experiment, estimate the expression of Cx43 and be assembled into patch through immunofluorescence staining.Operate of Zhang (1992) basically.In brief, (Nalge Nunc International, Naperville IL) go up growth, and with following agent treated 4 days: (1) is dissolved in 1 to the fusion culture of C3H/10T1/2 cell: 2EtOH/H at Permanox plastics 4 Room slides ₂The disodium salt disuccinic acid ester astaxanthin derivatives (the statistics mixture of steric isomer) of O prescription; (2) concentration in THF is 1 * 10 ^-8The retinoid TTNPB of M is as positive control; Or (3) 1: 2EtOH/H ₂O is as solvent control.With-20 ℃ of methyl alcohol cell fixation is spent the night,, be used in 1% bovine serum albumin (Sigma among the PBS with the damping fluid washing; St, Louis, MO) sealing; As (Zymed, San Francisco CA) cultivate with the anti-Cx43 antibody of rabbit polyclonal above (2); And with the anti-rabbit that combines Alexa568 two anti-(MolecularProbes, Eugene, OR) detections.With 568nm rayed slide, and under the 600nm wavelength, use Zeiss Axioscope opticmicroscope and Roper Scientific refrigerative CCD camera to obtain image.

Using contrast of TTNPB retinoid and concentration is 1 * 10 ^-5The slide of the statistics mixture process of the steric isomer of the disodium salt disuccinic acid ester astaxanthin derivatives of M shows immunoreactivity Cx43 and is being assembled into patch with cytolemma zone that flanking cell directly contacts.The position of the patch that this assembling and gap connect with form consistently, known said patch is that a plurality of individual gaps connect (Perkins, 1997) of assembling formation in the cell mass that correlates through connection.In the culture of the solvent treatment that is used as contrast; This immunoreation patch is uncommon, and than little at detected patch with the statistics mixture of the steric isomer of disodium salt disuccinic acid ester astaxanthin derivatives or in as the cell of the TTNPB processing of positive control.The frequency of these patches and size thereof are consistent with the function difference (the statistics mixture＞solvent control of the steric isomer of TTNPB＞disodium salt disuccinic acid ester astaxanthin) that is connected permeability in part 1 with the detected gap of the described fluorescent yellow dye shift experiment of Figure 14, and induce degree consistent with the Cx43 that in part 2 and the described immunoblot experiment of Figure 15, is detected.Representative Photomicrograph is shown in figure 16.

The inhibition that non-esterified free astaxanthin transforms carcinogens inductive tumour in the mouse fibroblast cell:

After the astaxanthin of oral esterification, in mammal intestine, produce non-esterified free astaxanthin.Only free astaxanthin is found in mammalian plasma and solid organ.This obtains proof once more in single and multi-dose oral pharmacokinetic; The result is as described herein.The parenteral administration disodium disuccinic acid ester astaxanthin derivatives (XVI) that acts on that mixes the esterase of dwelling in sero-abluminous intrinsic esterase activity and serum and the solid organ produces non-esterified free astaxanthin afterwards apace.

The flash photolysis experiment proves that also disodium disuccinic acid ester astaxanthin derivatives has identical anti-oxidant behavior with non-esterified free astaxanthin aspect the formation of carrotenoid radical cation.Experimentize and estimate non-esterified free astaxanthin (final split product in the body of disodium salt disuccinic acid ester astaxanthin derivatives (XVI); Steric isomer 3S as 1: 2: 1 ratio; 3S ', meso and 3R; The alltrans mixture test of 3 ' R) suppresses the ability that tumour transforms in the C3H10T1/2 cell culture model of in the laboratory of late CharlesHeidelberger, developing (Reznikoff, 1973).This cell culture system demonstrates the initiation and the transformation event (Bertram, 1985) of simulating effectively in full animal tumor forms.In these cells, carinogenicity polynuclear hydrocarbon 3-MECA (MCA) is handled in the cell of few partially disposed and is produced firing event, after 5 weeks, causes these cell generation form transformation, shows as to have the focus that transforms.These cell transformed are injected to the homology mouse cause forming sarcoma, show the carcinogenic character (Reznikoff, 1973) of conversion in the injection site.This test is transform as is suitable for detecting cancer prevention agent (Bertram, 1989), and cancer prevention property retinoid and carrotenoid have proved conversion (Bertram, 1991 that can suppress in this system; Pung, 1988; And Merriman, 1979).

The previous scheme of establishing of this experimental basis (Bertram, 1991 and Pung, 1988) is carried out.In brief, with the 10T that is derived from MEC 1/2 cell with 10 ³The density of cell/60mm plate be seeded in replenish 4% foetal calf serum (Atlanta Biologicals, Atlanta is GA) with 25 μ g/mL GT (Sigma; St.Louis; MO) Eagle minimum medium (BME) (Atlanta Biologicals, Atlanta, GA) in.(Sigma, St.Louis MO) or as 0.5% acetone (ultimate density) of contrast handle cell to be used in 5.0 μ g/mL MCA in the acetone after 24 hours.Handle back 24 hours replacing substratum at MCA.The astaxanthin or the retinol acetate in acetone that are used in after 7 days among the THF are handled cell, and handle in per 7 days again, continue for 4 weeks.With other plate of The suitable solvent control treatment.After experiment 5 weeks of beginning, use the methyl alcohol fixed cell, and (Sigma, St.Louis dye MO), and give II type and III type focus scoring (Reznikoff, 1973) with 10% Giemsa stain.

The result of this analysis shows with comparing with the cell of handling as the THF of solvent control with MCA to handle with astaxanthin and caused the MCA inductive to transform focus quantity generation concentration dependent in 4 weeks reducing (of Figure 34).Figure 34 describes effect non-esterified, that free astaxanthin (as the alltrans mixture of steric isomer) transforms MCA inductive tumour.The figure representative is 68 parts of usefulness 3 * 10 altogether ^-6M, 1 * 10 ^-6M and 3 * 10 ^-7The culture that the astaxanthin of M is handled, they are sent in 0.3%, 0.1% and 0.03% THF carrier respectively.Contrast as follows: 16 plates are not accepted carcinogens altogether, and handle with 0.05% alcohol solvent; Contrast does not show any transformation event.20 plates produce the transformation efficiency of 0.92 focus/plate with MCA and 1%THF solvent treatment altogether.The conversion of relatively calculating the plate that astaxanthin handles with the average focus/plate of the various processing of the contrast of handling with MCA-reduces percentage (% reduction).Use paired Student ' s t check to carry out reasoning property statistics, the P value that obtains calculating respectively is 0.00004,0.00001 and 0.00006.Think P＜0.05th, significant.With 3 * 10 ^-6The M astaxanthin is handled and to be caused suppressing fully the phenotype (Figure 35) that transforms.Figure 35 describes astaxanthin plate of handling and the comparison that contrasts plate.Representative plate is used following agent treated: A, no MCA, solvent control; B, MCA 5.0 μ g/mL, 1%THF is as solvent control; C, MCA, in THF 3 * 10 ^-6M astaxanthin (as the alltrans mixture of steric isomer).It should be noted that this inhibition level is considerably beyond the level of before having reported about all other carrotenoid that use same approach (Bertram, 1991) test.More present data with before transformed and reduce the data that percentage reports and show being tried tumour under the concentration, astaxanthin be than β-Hu Luobusu or Food Orange 8 effective the conversion inhibitors (Figure 36) of Duoing.Figure 36 describes the comparison that the carrotenoid of astaxanthin (as the mixture of steric isomer) and first Pretesting carries out.Editing data; The reduction percentage that MCA-inductive tumour transforms focus/plate in the culture that will handle with astaxanthin compares with the reduction percentage at the focus/plate that adopts identical scheme processing data afterwards to draw with β-Hu Luobusu and Food Orange 8 (Bertram, 1991) of previous Bertram laboratory report.The maximum concentration of formerly testing about β-Hu Luobusu and Food Orange 8 at this report (1 * 10 ^-5M) the reduction percentage under; Astaxanthin is not adopted this greater concn, because the activity that astaxanthin is measured is bigger under low concentration.These researchs show that the cracked astaxanthin part of synthesis of derivatives is the potential of highly effective cancer chemopreventive agent after oral and administered parenterally.Liver in conjunction with this paper also reports is accumulated pharmacokinetic data (after single and multiple doses strategy), and this application of compound constitutes useful especially embodiment.

Suppress reactive oxygen species:

In experiment, from people volunteer's whole blood, separate neutrophil with the Percoll gradient.Then isolating neutrophil is resuspended in the phosphate buffered saline (PBS), and does maximal stimulation to induce respiratory burst and to produce superoxide anion with phorbol ester.The disodium salt disuccinic acid ester astaxanthin derivatives that adds different concns in the solution of activatory human neutrophils is measured super-oxide signal [as with EPR (EPR) imaging mensuration] then.Disodium salt disuccinic acid ester astaxanthin derivatives (as the mixture of steric isomer) reduces the superoxide anion signal (Fig. 2) of mensuration with the mode that depends on dosage; Under 3mM concentration, realize being close to suppressing the superoxide anion signal fully.Fig. 2 is illustrated in the control group intensive super-oxide signal after the activation, and with the titrating result of disodium salt disuccinic acid ester astaxanthin derivatives of 100 μ M-3mM.The disodium salt disuccinic acid ester astaxanthin derivatives of test is removed 28% resultant signal under 100 μ M.Under 3mM, almost there is not super-oxide signal residue.These results show that the cardioprotection in ischemia-reperfusion injury that other above-mentioned anti-neutrophil intervention is confirmed also can use novel carotenoid verivate as herein described to realize.Except reducing superoxide anion signal important in ischemia-reperfusion injury; Possibly realize also that with described novel carotenoid verivate cardiac muscle rescues, because superoxide anion plays a major role between the extended period at myocardial ischaemia in tissue injury and death.

Fig. 3 describes the effect of disodium salt disuccinic acid ester astaxanthin derivatives/vitamin c solution to reactive oxygen species (superoxide anion), and this is with EPR imaging monitoring.Solution comprises about 2 respectively than about 1 the vitamins C and the mixture of disodium salt disuccinic acid ester astaxanthin derivatives.Disodium salt disuccinic acid ester astaxanthin derivatives/vitamin c solution reduces the superoxide anion signal (Fig. 3) of mensuration with the mode that depends on dosage; Realize suppressing fully the superoxide anion signal in 0.02 μ M concentration.Fig. 3 proof is the strong super-oxide signal after the activation in control group, and with the titrating result of disodium salt disuccinic acid ester astaxanthin derivatives/vitamin c solution of 0.01 μ M-0.02 μ M.

In the 3rd experiment, from the second people volunteer's whole blood, separate neutrophil with the Percoll gradient once more.Then isolating neutrophil is resuspended in the phosphate buffered saline (PBS), and does maximal stimulation to induce respiratory burst and to produce superoxide anion with phorbol ester.The solution of past activatory human neutrophils adds the hydrochloride two Methionin ester astaxanthin derivatives (XX) of 4 kinds of concentration, measures super-oxide signal (measuring with the EPR imaging) then.Hydrochloride two Methionin ester astaxanthin derivatives also reduce the superoxide anion signal (Figure 21) of mensuration with the mode that depends on dosage, under 1 μ M, reducing about 5% under 3mM, reducing 98%.Under 3mM concentration, realize near fully suppressing the superoxide anion signal once more.This new carotenoid derivatives shows to remove under lower concentration (1 μ M) and renders a service, and this verivate approaching ability of fully eliminating the superoxide anion signal in this in vitro tests that increases concentration.

This novel derivative shows novel derivative (disodium disuccinic acid ester astaxanthin once more in external activity as the water-based scavenging agent; Hydrochloride two Methionin astaxanthins) will serve as soft medicine (promptly activity being arranged) rather than prodrug (not having activity) in vivo until being cracked into free astaxanthin as complete, uncracked novel derivative.This verivate (XX) water-soluble greater than 50mg/mL shows that the inventive method can be used for increasing the water-soluble of parent carrotenoid (being astaxanthin in the case), near zero the paramount mg/mL scope of intrinsic water-soluble increase.

The direct superoxide anion that disodium disuccinic acid ester astaxanthin derivatives causes is removed: each steric isomer that shows through the EPR imaging method is with respect to the relative effectivenes of the statistics mixture of steric isomer

Material

Non-esterified full E astaxanthin [steric isomer 3S, 3 ' S, meso (3S, 3 ' R and 3 ' S, 3R) and 3R, and 1: 2: 1 statistics mixture of 3 ' R] available from Buckton Scott (India), and use (HPLC shows＞95% purity) as providing.Astaxanthin is dissolved in HPLC level methyl-sulphoxide (DMSO; Sigma-Aldrich, St.Louis, MO).Test the disodium disuccinic acid ester derivative of astaxanthin respectively separately with 9 kinds of prescriptions: the statistics mixture of steric isomer (about astaxanthin, above-mentioned, 1: 2: 1 mixture of full E; In all tables and figure, be labeled as " mixture "); 3S, 3 ' S and 3R, 3 ' R (optical isomer or enantiomer); And meso (3S, 3 ' R and 3 ' S, the mixture of 3R; The diastereomer that enantiomer is right).The synthetic purity of all disuccinic acid ester derivatives is according to HPLC＞90%.At first under the ultimate density that in pure water solution (deionized water), suits, test the disuccinic acid ester derivative from the 10mM stock solution.Then by in 1: 2 mixture of alcoholic acid with the stock solution (ultimate density 33 of EtOH in the stock solution of 10mM preparation ¹/ ₃%; Ultimate density 0.3% in the isolating neutrophil test; HPLC level ethanol, Sigma-Aldrich, St.Louis, MO) each in 4 kinds of disuccinic acid ester derivatives of test.Also test 3S, 3 ' S verivate by the stock solution (ultimate density 0.5% in isolating neutrophil test) of 50%EtOH concentration.The ethanol formula table of disuccinic acid ester derivative reveals the supramolecular assemblies that complete depolymerization forms in pure water solution, the monomer solution of verivate is provided, and introduces test then immediately.Also carry out independent alcoholic acid negative control (is 0.3% and 0.5% final EtOH concentration) and superoxide dismutase mimetic positive control (10 μ M ultimate densities in isolating neutrophil test;

Pharmaceuticals; Inc.; St.Louis, MO).

Synthetic carotenoid derivatives [succsinic acid one-(4-{18-[4-(3-carboxyl-propionyloxy)-2,6,6-trimethylammonium-3-oxo-hexamethylene-1-thiazolinyl]-3,7; 12,16-tetramethyl--octadecane-1,3,5; 7,9,11,13; 15,17-nonene base }-3,5,5-trimethylammonium-2-oxo-hexamethylene-3-thiazolinyl) ester; Figure 17] and stereoisomeric forms in any ratio, the disodium disuccinic acid ester derivative of astaxanthin, alltrans is (complete-E) form.Said verivate for 3 and 3 ' carbon location have the symmetrical chiral molecules of 2 chiral centres, comprise 4 kinds of steric isomer: 3R, 3 ' R and 3S; 3 ' S (optical isomer or enantiomer); And the diastereomer meso-form (3R, 3 ' S and 3 ' R, 3S).Comprise the 3R of 1: 2: 1 ratio by the statistics mixture of the astaxanthin synthetic steric isomer of commercial source, and 3 ' R, meso (3R, 3 ' S and 3 ' R, 3S) and 3S, 3 ' S stereoisomeric forms in any ratio.All independent steric isomers and statistics mixture synthesize purity＞90% according to HPLC, allow directly the directly individuality effectiveness of free-radical scavengers of relatively these forms conducts.Complete-E form the steric isomer that is used for this research is linear, stiff molecule (bolaamphiphiles), because in the polyenoid chain of spacer, lack cis (or Z) configuration.

The disodium disuccinic acid diester of astaxanthin increases with respect to parent compound astaxanthin performance water outlet " dispersiveness ".The water dispersible of each steric isomer and statistics mixture makes them can be introduced into the buffering water-based test system that does not contain solubility promoter all greater than 8mg/mL (approximately 10mM).Verivate for test in this research is also observed parent carrotenoid such as astaxanthin (Salares; 1977) and novel carotenoid verivate (for example capsanthin verivate) (Zsila; 2001 and Bikadi, 2002) in the aqueous solution, form the trend of supramolecular assemblies.

Supramolecule oneself assembling causes sizable aggregate in the aqueous solution, and stops the maximum direct interaction of assembling molecule and radical kind.Therefore, at the water-based prescription with contain that the direct removing of newer astaxanthin derivatives shows in the solubility promoter alcoholic acid prescription.In stock solution, the EtOH/ water meter of 1: 2 concentration reveals complete depolymerization statistics mixture, meso and 3R, 3 ' R verivate; Need 50% ethanol stock solution to come complete depolymerization 3S, 3 ' S isomer.Also with respect to negative (being the ethanol carrier) removing ability with positive [superoxide-dismutase (SOD) stand-in, the racemize astaxanthin that dissociates is in DMSO] blank determination compound.

White corpuscle purifying and preparation

Through Percoll density gradient centrifugation separation of human polymorphonuclear leukocyte (PMNs) from the single volunteer's of fresh sampling venous blood (S.F.L.), producing PMN purity is＞95%.

Each 10mL whole blood is mixed with 0.8mL 0.1M EDTA and 25mL salt solution.The blood of dilution is laid on the 9mL Percoll of 1.080g/mL specific density.At 400 * g with after 20 ℃ times centrifugal 20 minutes, shift out blood plasma, monocyte and Percoll layer.Through adding the 18mL icy water 30 seconds, add then 2mL 10 * PIPES damping fluid (25mM PIPES, 110mM NaCl and 5mM KCl, with the NaOH titration to pH 7.4) lysed erythrocyte.Under 4 ℃ with cell precipitation, supernatant decanted liquid, and repeat this process.After second time hypotonic dissolution, with twice of PAG damping fluid (the PIPES damping fluid comprises 0.003% human serum albumin and 0.1% glucose) washed cell.Then, count PMN through on hematimeter, carrying out light microscopy.(the PAG damping fluid contains 1mM CaCl then final deposition to be suspended in the PAG-CM damping fluid ₂With 1mM MgCl ₂) in.

EPR measures

All EPR measure and use at the X-bands of a spectrum with TM ₁₁₀The Bruker ER 300 EPR spectrometers of chamber operation carry out.(San Jose CA) measures microwave frequency for EIP Microwave, Inc. with Model 575 microwave telltales.For measuring the PMN generation O that phorbol-ester (PMA) stimulates ^- ₂, (Oxis, Portland OR) carry out EPR spin-capture research with the DEPMPO of 10mM.Stimulate 1 * 10 with PMA (1ng/mL) ⁶PMNs, and it is filled to kapillary measures to carry out EPR.For being determined at non-esterified free " racemize " astaxanthin and the radical scavenging activity of the disodium salt disuccinic acid ester derivative in every kind of 9 kinds of prescriptions among the DMSO,, carry out the PMA stimulation as aforementioned then with PMN and compound preincubation 5 minutes.The instrument of testing that is used to spin-capture is provided with as follows: the amplitude of accommodation, 0.32G; Time constant, 0.16s; Sweep time, 60s; Regulating frequency, 100kHz; Microwave power, 20 milliwatts; And microwave frequency, 9.76GHz.Sample is placed quartzy EPR flat chamber, and spectra re-recorded.As identify the composition signal in the spectrum reporting and carry out quantitatively (Lee, 2000).

Statistical study

(NCSS 2001 and PASS 2002, Kaysville UT) carries out statistical study with the NCSS statistical package.All statistical test time are carried out in α=0.05.

EPR result's summary:

Will be when the research beginning by Metaphore, effective SOD stand-in that Inc. produces are as positive control.As observed repeatedly in the Zweier laboratory, 10 μ M dosage in water carrier only are close to eliminates the superoxide anion signal fully, and (97% suppresses as detecting with DEPMPO; Table 1).Also estimated independent alcoholic acid negative control (ultimate density 0.3%), because ethanol shows less removing activity in these systems; Under this concentration, see 5.7% restraining effect.In the data of description of table 1, from comprise the alcoholic acid prescription, do not deduct this amount of suppression, because in this research, estimate the effectiveness of drug administration carrier (the disodium disuccinic acid ester derivative in EtOH) in directly removing itself.The reference standard that the non-esterified free astaxanthin (100 μ M) of evaluation in DMSO directly compares as synthetic novel derivative in studying therewith; The average restraining effect of astaxanthin/DMSO carrier is 28% (table 1).

Figure 18 shows that ultimate density is each a relative removing ability in 4 kinds of steric isomers in water (mixture and 3 kinds of independent steric isomers) of 100 μ M.Except 3R, 3 ' R enantiomer (28.7% suppress), all other novel derivative formula tables reveal that (scope-2.0% suppresses to 19.3% with respect to the removing ability that astaxanthin/DMSO prescription reduces; Table 1).Can find out, 3S, it is active that 3 ' S prescription does not show any average removing.But, in the time of in the ethanol prescription, being incorporated into isolating neutrophil test system, removing active the increase in each case and surpass the identical verivate (Figure 19 that in water, prepares; Scope 38.0%-42.5%).Be important to note that 3S, 3 ' S verivate is prepared in 50%EtOH to carry out this comparison.The novel derivative removing ability of observing in the ethanol prescription increases the trend that surpasses the astaxanthin in DMSO, but after the average removing ability of deduction ethanol carrier (ultimate density in test is 0.3%), this trend not obvious (NS).In addition, do not observe the significant difference (Figure 19) of average removing ability between the novel derivative preparation that 4 kinds in ethanol are tried.

Figure 20 shows that the mixture of steric isomer of the disodium disuccinic acid ester astaxanthin in the ethanol prescription that increases concentration is to the titration results of super-oxide signal suppressing.When concentration when 100 μ M increase to 3mM, observing the super-oxide signal (is 95.0% to suppress under 3mM dosage near the complete inhibition effect; Table 1 and Figure 18).Dose-response curve is non-linear.Adjust suppressing percentage and proof load, disodium disuccinic acid ester derivative is used as low 1 to 2 one magnitude (table 1) of effectiveness of the SOD stand-in of positive control than in this research.Table 1 is described in the descriptive statistics of the different ingredients of the disodium disuccinic acid ester derivative of the astaxanthin of test in this research.The sample size that each prescription is estimated is 3 or bigger, except the 3S in the 50%EtOH stock solution, the SOD stand-in of 3 ' S (N=2) and evaluation when the research beginning (positive control, N=1).

Table 1

EPR result's summary

Astaxanthin is a kind of effective lipophilic antioxidant, and it is performance its antioxidant property (Britton, 1995) in being rich in the cytolemma of lipid, lipoprotein and other tissue usually.The verivate of the astaxanthin that increases as water dispersible promoting agent effectiveness has the ability of direct removing by the water superoxide anion of isolating human neutrophils generation after respiratory burst stimulates.

The water-based prescription of novel derivative in the effectiveness of directly removing ability to less than the ethanol prescription.The assembling of the supramolecule of water-soluble carotenoid verivate can be explained the shortage that they are renderd a service in these solvents in some solvent (for example water).Said gathering is spirrillum, " card-pack " type, in water-based solution, forms the aggregation that size surpasses 240nm.The ionic strength that increases damping fluid can increase the big or small and stable of these aggregates.The radical scavenging activity of these aggregates is compared with the monomer solution of same compound and is reduced; In fact, for water-soluble 3S, 3 ' S steric isomer do not see the removing ability (table 1, Figure 18).Preparation is used for external and prescription in vivo tests with taking every caution against error because supramolecule assembling restriction can with the quantity of the interactional molecule of radical kind.The size of aggregate is also necessary to be considered, has described to comprise as many as 10 ⁶Individual molecule and size reach 300nm or bigger aggregate (Bakadi, 2002).

Titration disodium disuccinic acid ester astaxanthin derivatives dosage to 3mM (as 1: the mixture of the steric isomer in the 2EtOH/ water) show near suppressing superoxide anion signal (95% suppresses) fully, as measuring (Figure 20) with DEPMPO spin hydrazine.Dose-response curve is non-linear, requires to increase dosage with the approaching radical signal (Figure 20) that suppresses fully.Under minimum test concentrations (100 μ M), suppress nearly 40% signal.The effectiveness of disodium disuccinic acid ester astaxanthin derivatives can directly compare with superoxide-dismutase (SOD) stand-in that are used as the positive control in this research (is 97% inhibition at 10 μ M) under this dosage.These results show that as the water free-radical scavengers, disodium disuccinic acid ester astaxanthin derivatives is than little 1 to 2 one magnitude of effectiveness of SOD stand-in.But; These verivates decay into free astaxanthin in vivo; It [comprises mitochondrial membrane and nuclear membrane (Goto being rich in the cytolemma of lipid; 2001) becoming] has activity, thereby double protection (water and fat free radical scavenging mutually) is provided, and this is irrealizable with water-soluble protein and enzyme simulation thing.Non-esterified free astaxanthin (when providing as diet supplement with 0.02% food wt/wt) has the cardioprotection to the arduous motion infringement of Skelettmuskel and cardiac muscle people 2003 such as () Aoi of anti-ROS mediation.Therefore, this compounds can be extraly prevented as radical and reactive oxygen species is the clinical treatment agent (Cross, 1987) in the important indication to this characteristic (being two-phase free radical scavenging).

This research proves by the direct removing of the detected one type of new carotenoid derivatives of EPR spectroscopy to superoxide anion for the first time.Find that said compound forms supramolecular assemblies in pure water solution.With respect to the monomer solution of same compound, the formation of supramolecular assemblies possibly limit their removing and render a service.In 4 kinds of potential steric isomers of said new compound, do not see the significant difference of removing ability.Dosage-scope research shows that the concentration of verivate can increase near suppressing inductive superoxide anion signal fully.As the agent of potential interior therapeutic, this compounds can have the acute and chronic disease of attested effectiveness thereby can be widely used for effective free-radical scavengers to it as water and fat scavenging agent mutually.The direct superoxide anion that disodium disuccinic acid ester two vitamins C astaxanthin derivatives cause is removed:

In EPR (EPR) imaging experiment, from people volunteer's whole blood, separate neutrophil with the Percoll gradient.Then isolating neutrophil is resuspended in the phosphate buffered saline (PBS), and does maximal stimulation to induce respiratory burst and to produce superoxide anion with phorbol ester.The disodium disuccinic acid ester two vitamins C astaxanthin derivatives (XXIII) of the solution adding different concns of past activatory human neutrophils (half systematic naming method succsinic acid 4-[18-(4-{3-[2-(3,4-dihydroxyl-5-oxo-2,5-dihydrofuran--2-yl)-2-hydroxyl-ethoxy carbonyl]-propionyloxy }-2,6,6-trimethylammonium-2-oxo-hexamethylene-1-thiazolinyl)-3; 7,12,16-tetramethyl--octadecane-1,3; 5,7,9,11; 13,15,17-nonene base]-3,5; 5-trimethylammonium-2-oxo-hexamethylene-3-alkenyl esters 2-(3,4-dihydroxyl-5-oxo-2,5-dihydrofuran--2-yl)-2-hydroxyl-ethyl ester), measure super-oxide signal (as measuring) then with the EPR imaging.Disodium disuccinic acid ester two vitamins C astaxanthin derivatives (XXIII) reduce the superoxide anion signal (Figure 33) of mensuration with the mode that depends on dosage; Under 60 μ M concentration, realize suppressing fully the superoxide anion signal.This representative is renderd a service than the same synthetic disodium disuccinic acid ester astaxanthin derivatives (XVI) that is used for this serial experiment increases by 50 times.The purity of the verivate of test is 88% (confirming through HPLC TG-AUC or AUC).This new carotenoid derivatives-be designed to " soft medicine "-keep anti-oxidant function of independent vitamins C molecule through each ascorbic 6-OH position being carried out esterification.The effectiveness of verivate (XXIII) is near the effectiveness (it suppresses the superoxide anion signal fully with 20 μ M/40 μ M disodium disuccinic acid ester astaxanthin derivatives (XVI)/free vitamin C prescription) of the prescription of the disodium disuccinic acid ester astaxanthin (XVI) of 1: 2 mol ratio and free vitamin C.The verivate (XXIII) that every mole verivate produces 2 moles of free vitamin C and 1 mole of non-esterified free astaxanthin in vivo is particularly preferred for some clinical indication.Verivate (XXIII) also possibly show under these clinical settings and render a service increase: wherein water is removed (through complete parent verivate and free vitamin C) to remove (through non-esterified free astaxanthin) mutually is important with fat for the minimizing that is attributable to the pathologic condition that ROS and other radical kind damage.

Infraction size in the male Sprague-Dawley rat reduces:

Fig. 4, Figure 25 and Figure 26 describe big or small the reducing of infraction in the male Sprague-Dawley rat.Be used in disodium salt disuccinic acid ester astaxanthin derivatives (as the mixture of steric isomer) the pre-treatment male Sprague-Dawley rat in the aqueous solution, carry out obturation then and induce myocardial infarction.With 100mg/kg Inactin anesthesia male Sprague-Dawley rat (175-200 restrains s), carry out instrumentation, and expose heart.Around arteria coroaria sinistra placed around suture, and carry out total coronary occlusion of 30 minutes, carry out perfusion again in 2 hours then, cut off heart from animal and measure the infraction size this moment.Wash heart with damping fluid, and cultivate with triphenyltetrazolium father-in-law muriate (TTC) dyeing solution in the phosphate buffered saline buffer of pH 7.40 that remains under 37 ℃.Infraction size (IS) be expressed as the % that accounts for dangerous area (IS/AAR, %).Monitoring systems blood pressure, heart rate, vim and vigour and body temperature in whole experiment, and body temperature and vim and vigour is strict controlled in the normal physiological level.Through disodium salt disuccinic acid ester astaxanthin derivatives or the Sterile Saline carrier of tail vein injection I.V. administration 25,50 4 day by a definite date every day or 75mg/kg, block experiment then and measure the infraction size.

Rescue result summary.

The infraction size reduces and corresponding cardiac muscle rescue is linear and increases (P=0.001**) significantly with dosage.Receiving greatly most under the amount of reagent 75mg/kg, it is 56% that mean myocardial is rescued, and it is approaching with the obtainable rescue rate of local asphyxia pretreatment strategy.Before testing more high dosage, volume restrictions is arranged in this rat, carrying out single dose I.V. injection; But the significant linear dependence (P＜0.001** between the astaxanthin of non-esterified free blood plasma level and the IS/AAR%; r ²=0.67) is illustrated in and realizes under the dosage of about 120-125mg/kg that 100% rescues.This proves the cardioprotection of novel carotenoid verivate for the first time.

The target tissue of the pharmacokinetics of oral disodium disuccinic acid ester astaxanthin derivatives, the bioavailability of increase and increase distributes:

The blood plasma pharmacokinetics

The oral pharmacokinetic parameter of single dose of on male C57BL/6 mouse, measuring disodium disuccinic acid ester astaxanthin derivatives (comprises C _Max, T _Max, AUC _(0-72)V _dAnd clearance rate).Can effectively prevent CCl in the Sprague-Dawley rat to demonstrate in the research formerly ₄The single maximal dose (500mg/kg) of the damage of secondary after the administration (in these researchs is the 100mg/kg body weight) is given the oral verivate of animal.At following time point, obtain to be used for the dissociate sample of astaxanthin level of HPLC analysed for plasma and liver from least 3 animals of each time point:

Time 0 [before the test-compound administration, carrying out immediately], absorption back 2,4,6,8,12,16,24,48 and 72 hours.

With other (10,14 and 36 hours at interval; Table 2 and 3) gets the additional sample of N＜3.In this research, in mammal intestine, be cracked into dissociation Serlabo fully when ester of carotenoid, it is passive measures non-esterified free astaxanthin level when moving through intestinal cells.

Experimental technique summary: blood plasma pharmacokinetics

With male C57BL/6 mouse, approximately 25g closes and supports in cage (3 mouse/cages), and ad lib standard mouse food (Purina Mouse Chow, Ralston Purina St.Louis) and water, continue at least 5 days, begin then to test.Disodium disuccinic acid ester astaxanthin derivatives is mixed the emulsion that is suitable for the per os tube feed with preparation with following component:

● sterile filtration (0.2 micron

) water;

● sweet oil (Bertolli USA, Inc., Secaucus, NJ);

● soybean lecithin, IV-S type (Sigma-Aldrich Co., St.Louis, MO; Catalog number (Cat.No.) P3644).

Disodium disuccinic acid ester astaxanthin derivatives shows the water-soluble of about 8.64mg/mL in the water-based prescription.In above-mentioned emulsion, solubleness increases to about 50mg/mL, allows in these animals through the gavage nearly 500mg/kg that takes medicine.This significantly 6 times of gavage researchs that are beneficial to these mouse greatly of increase of solubleness in drug administration carrier.

The method that is used to prepare emulsion is following:

(1) 80mg soybean lecithin (Sigma catalog P3644) is added to 5.0mL water.About 30 minutes of vortex mixed is until the suspension-s homogeneous on 15mL centrifuge tube discontinuous ground;

(2) add 2.5mL sweet oil and vortex mixed under the room temperature.This produces the yellow suspension of homogeneous, stiff, muddiness.This emulsion material can store in room temperature or 4 ℃ of refrigerators.If store, vortex mixed at once before the 3rd step (following) added disodium disuccinic acid ester derivative;

(3) the disodium disuccinic acid ester astaxanthin derivatives with 50mg/mL directly is added to emulsion.This compound easily gets into homogeneous suspension-s under this concentration.Vortex mixed is carried out tube feed then immediately to guarantee homogeneous suspension-s; With

(4) said material has the possibility of stopping up mouse tube feed pin.After per 2 tube feed, wash the tube feed pin.

Use emulsion through oral gavage with 500mg/kg body weight single dose.Withdraw from food the evening before experiment from all cages.After using emulsion 1 hour, all animals are recovered food and water.

The method that is used for whole blood and sample of tissue, sample extraction and HPLC analysis has been made detailed description (Osterlie, 2000).In brief; With whole blood collection comprise EDTA

pipe in; Then through at 4 ℃, prepared blood plasma under 1500 * g in centrifugal 20 minutes.Subsequently with the plasma sample aliquots containigization, and in liquid nitrogen quick freezing, transport then and carry out HPLC and analyze.

Tissue accumulation

The free astaxanthin concentration in the time point determining liver identical also with plasma sample.Put to death the back and from each animal of pharmacokinetic, take out liver, and in liquid nitrogen quick freezing.Be used for the hepatic tissue that HPLC analyzes like said (Jewell, 1999) preparation.Therefore, check simultaneously that at the time point identical the liver of free astaxanthin accumulates with plasma analysis.

The experimental technique summary: the liver of free astaxanthin is accumulated

Quick freezing is from the liver of 300mg at the most of each animal in liquid nitrogen.With the mixture of chloroform/methanol/water, carry out tissue homogenate and extraction according to the method (1999) of Jewell.Accumulate through non-esterified free astaxanthin in the HPLC method evaluation liver about plasma sample is said as above then.

Pharmacokinetics result's summary

The conclusive table of putting down at the blood plasma of the free astaxanthin of suitable sampling interval time and edema due to dysfunction of the liver is shown in table 2 and 3.The free astaxanthin TG-AUC vs. time (AUC ' s) be also included within the table 2 and 3 that blood plasma and liver are non-esterified.These results show, for each sampling interval, free astaxanthin level is identical or greater than the level in blood plasma in the liver.It is beyond example in document that the tissue specificity of this improvement is delivered to liver; In fact the edema due to dysfunction of the liver of free astaxanthin is put down and is usually less than the respective horizontal in the identical time point blood plasma (Kurihara, 2002) after administration.Therefore, the disodium disuccinic acid ester astaxanthin derivatives in above-mentioned emulsion is to be used in the oral excellent carrier of dissociation Serlabo to the tissue of interest of delivery treatments concentration afterwards.

Table 2: the blood plasma level of non-esterified free astaxanthin

Table 3: the edema due to dysfunction of the liver of non-esterified free astaxanthin is flat

When in oral carrier or food, carrotenoid such as astaxanthin being provided, need carry out pre-treatment (15 days to 6 weeks) usually in liver injury research, to realize level of significance (Kang, 2001; Kim, 1997; People such as Aoi 1993).In the case, realize treatment level (200nm or more than) with single dose.

0.9mg/L C _Max(table 4) also is unprecedented in rodent, and these animals only absorb the carrotenoid of the oral dosage of little per-cent.These blood plasma of dissociation Serlabo and edema due to dysfunction of the liver are flat to be after the single dose compound of only taking in emulsion carriers, just to obtain, and this point is significant.At philtrum, people such as Osterlie (2000) are described in the C after the non-esterified free astaxanthin of single dose 100mg (approximately 1.1mg/kg oral dosage) in the sweet oil carrier _MaxBlood plasma level is 1.3mg/L.The people absorbs the carrotenoid of 40-50% oral dosage usually when in fat carrier, providing, and rodent has only a few percentage point by contrast.Therefore, this research proof is used emulsion carriers as the rodent development to reach the nearly 70% of philtrum Cmax, thereby is increased the availability that this verivate is used for liver protection research greatly.

Table 4:pK parameter

Parameter	Liver	Blood plasma
			*C max(mg/L) **T max(hr) eliminate the transformation period (hr) and eliminate speed (1/hr) * * * AUC (0-72) (mg hr/L) * * * AUC ∞ (mg hr/L) oral clearance (L/hr) volume of distribution (L/kg)	0.9 6 11.655 0.059 15.8 15.9 15.856 263.9	0.2 6 3.938 0.176 1.2 1.2 216.822 1232.1

* peak concentration

The time of * peak concentration

* * TG-AUC

Parenteral administration Cardax ^TM(disodium disuccinic acid ester astaxanthin derivatives) reduction of the cyclical level of experimental infraction size and C-reactive protein in the rabbit afterwards:

With people such as Barrett people's such as (2002) method and carry out trickle modification and study parenteral administration disodium disuccinic acid ester astaxanthin derivatives (XVI) inductive infraction size in the rabbit is reduced the influence with inductive circulation C-reactive protein (CRP) level.The purpose of this research is to investigate the ability that disodium disuccinic acid ester astaxanthin derivatives (XVI) reduces inflammation, and this measures through CRP under experimental myocardial ischaemia/reperfusion injury situation in rabbit heart.Propose the CRP that makes the sign of acute inflammation (" acute phase ") reaction commonly used and possibly in fact have the short scorching effect that mediates through the complement activation cascade.Follow oxyradical (ROS) but form the myocardial ischaemia/reperfusion injury that increases and shown the complement activation system.Prove that in this system endogenous that (1) is secondary to the plasma C RP of far-end inflammatory lesions increases and be secondary to regional local asphyxia and the increase of dabbling myocardial tissue damage is relevant again; (2) increase of this damage (showing as the infraction size increases) is mediated by complement activity; (3) CRP is a kind of " effector, and be not only the indirect measurement of systemic inflammation.Therefore, circulation CRP level reduces and had before used Cardax ^TMBeing seen infraction size reduces together in rodent, forms the strong anti-inflammatory treatment therapy in the acute coronary syndrome situation.

In brief, male New Zealand rabbit (2.25-2.5kg) is used for this research.Induce the acute phase Inflammatory response through 1% Oleum Tiglii of 4 aliquots containigs of subcutaneous injection (each 0.5mL) in Semen Maydis oil, said injection starts from using Cardax ^TMCarried out pretreated second day.Use the once Cardax in water or isopyknic Sterile Saline every day ^TM(50mg/kg IV is through the ear vein injection) continued 4 days, accomplished experimental infarction then at the 5th day.Like aforementioned people 2002 such as () Barret, use method, with anti-rabbit CRP antibody obtain the circulating time course of CRP level increase based on ELISA.In the last day (the 5th day: medicine is inculcated the last time after about 24 hours) of experiment, use the mixture of xylazine (3mg/kg) and ketamine (35mg/kg), then intramuscular use Sodital (90mg/kg) anesthetized rabbit.Use Sodital when needing again to keep anesthesia.After tracheotomy, ventilate to rabbit with room air, and expose heart through left throacotomy.Then heart is bearing in the pericardium support, and 3-0 silk ligature is placed on around the LADCA.Through toward ligature is added tractive force with arterial occlusion 30 minutes, and then poured into 180 minutes.Before this scheme of completion, carve and promptly obtain venous samples can to measure plasma C RP.

When the flush phase again of this scheme is accomplished, take out heart, and on Langendorff perfusion instrument, insert conduit through aorta.Then with the Krebs-Henseleit damping fluid of improveing perfused hearts 10-15 minute again (20-25mL/ branch).When this section period finishes, use 80mL0.4%2,3,5-triphenyltetrazolium father-in-law muriate (TTC) 37 ℃ of following perfused hearts to confirm hazardous location (AAR).Then with surgery preparation/experiment infarction during identical regional ligation left circumflex artery.At this moment, stop filling pump, and through with the telescopic joint side mouth of aorta the blue dyestuff of 3.0mL Evan slowly being injected into heart.Make said solution be distributed to whole heart about 30 seconds.Meet at right angles with Z-axis then heart is cut into six lateral parts.Discard right ventricle, the apex of the heart and atrial tissue.The tissue representative of being demarcated by purple/blueness is by the relevant coronary artery of the non-infraction dabbling zone that distributes.Two surfaces of each crosscut part are printed to clean acetic ester sheet, it is scanned, digitizing is to calculate infarct size then.Total critical area(s) product representation is the percentage ratio of left ventricle.To block size then and be expressed as the percentage ratio that accounts for dangerous area.

Control animal and Cardax ^TMThe average infraction size of the animal of handling is shown in figure 37.Control animal and Cardax ^TMThe circulation CRP level of the animal of handling (being expressed as between baseline water and the mean difference between the inductive level when pouring into again) is shown in figure 38.At Cardax ^TMSee in the rabbit of handling that the infraction size reduces about 55.4%; Two groups local asphyxia hazardous location is similar.Similarly, the average increase (+23.5%) of circulation CRP level that exceeds baseline in the contrast is at Cardax ^TMEliminate fully in the animal of handling, to being lower than in the observed M.L. of baseline (15.7%).Since CRP is a effector in the acute coronary syndrome-under the situation that this acute phase reactant level raises, cause block size increase-be again that the reduction of the strong independence prediction thing-this circulating protein level of elementary and secondary prevention heart patient central vessel risk forms strong treatment therapy.

Oral disodium disuccinic acid ester astaxanthin reduces the alanine aminotransferase (ALT) that LPS (LPS) causes in the mouse and raises:

The effectiveness of the liver provide protection of the oral disodium disuccinic acid of following research evaluation ester astaxanthin derivatives in LPS inductive mouse liver injury model.

The experimental technique summary:

The male ICR mouse of handling for three monthly ages with LPS and GalN is to induce liver injury (Leist, 1995).At first as far as its mouse oral tube feed sweet oil/water/lecithin emulsion (10mL/kg or for 30 gram mouse 0.3mL) or comprise the identical emulsion of disodium disuccinic acid ester astaxanthin derivatives (50mg/mL), final disodium disuccinic acid ester astaxanthin dosage is 500mg/kg.After 2 hours to the solution of (IP) pump pickle (10mL/kg) or Escherichia coli LPS in the mouse peritoneum (3mg/kg, Sigma catalog number (Cat.No.) L-3755) and D-galactosamine (700mg/kg).IP injects back 5 hours with carbonic acid gas (CO ₂) smoothing method execution animal, collect blood plasma then and be used for ALT mensuration.

LPS inductive damage result's summary.

These PRELIMINARY RESULTS proof disodium disuccinic acid ester astaxanthin derivatives are to the not effect of plasma A LT of saline injection (the false processing of liver injury contrasts) animal.In the control animal of only using emulsion (not containing verivate) tube feed, ALT increases greater than 3 times.In the animal of the emulsion of accepting to contain 500mg/kg disodium disuccinic acid ester astaxanthin derivatives, ALT raises and reduces (N=3 animal/group) greatly, proves the effectiveness of compound reduction as the ALT of the serum mark of hepatic necrosis in these animals.Because the liver injury of LPS inductive is mediated by ROS (comprising free-radical oxidn nitrogen NO.); And significant system inflammation (non-esterified free astaxanthin has provide protection people 2003 such as () Ohgami to this) takes place in LPS infringement back, and this novel derivative is used for the effectiveness that this therein inflammation obtains promoted clinical indication and represents a useful especially embodiment.

Free astaxanthin accumulating in blood plasma and liver after the black mouse multi-dose oral:

In this pharmacokinetic, use method as herein described, give the disodium disuccinic acid ester astaxanthin derivatives (500mg/kg) of ten one (11) the individual independent day oral dosages of black its mouse oral tube feed in emulsion carriers, and at possible C _MaxAnd T _MaxFree astaxanthin accumulating in blood plasma and liver measured in (6 hours) in 3 animals.Possible C _MaxAnd T _Max(6 hours) are to be derived out by blood plasma in the oral pharmacokinetic of front single dose and liver sample.Evaluation non-esterified free astaxanthin accumulating in blood plasma and liver after single emulsion dosage.The mean plasma concentration of all animal subjects is 381nM.The average liver concentration of all animal subjects is 1735nM.In single dose research, on an average, in blood plasma and liver, all obtain the protection level and (non-esterified free astaxanthin is located at anti-oxidant ED ₅₀Be 200nM); The average liver concentration difference that is obtained seldom is 9 times of protection level.

In multiple doses research, peak and paddy level get all that (administration 6 hours afterwards is at possible C _MaxGet the peak level; At C _Max12 hours acquisition paddy levels after back 6 hours or the administration).Average peak level in Feng Hegu blood plasma is respectively 485nM and 231nM; Average peak level the Feng Hegu liver is respectively 1760nM and 519nM.Equally, under various situation, obtain the protection level, and be retained to behind the multiple dose administration 11 days; Under the situation of liver, obtain similar level for 9 times of protection levels.Equally, accumulating greater than observed accumulating in the blood plasma in each time point liver behind multiple dosing shows that this drug administration carrier is used for the effectiveness increase (Figure 32) of target to this solid organ.Data set finds out obviously that also long-term application disodium disuccinic acid ester astaxanthin derivatives will be effective aspect the liver protection thus.

Free astaxanthin accumulating in cardiac muscle (heart) and brain after black mouse single dose is oral:

The administered through oral gavage will be in emulsion carriers the disodium disuccinic acid ester astaxanthin derivatives (500mg/kg) of single maximal dose be applied to black mouse, and in 4 animals at possible C _MaxAnd T _MaxAccumulating of non-esterified free astaxanthin measured in (6 hours), and Cmax and Tmax derive according to blood plasma and liver sample in the research of front.Free astaxanthin non-esterified behind the single dose accumulating in heart be excellent (mean+/-SEM=693.25+ of 4 animals/-272nM), and with non-esterified free astaxanthin in liver to accumulate the result parallel.Equally, at each time point, accumulating greater than observed accumulating in the blood plasma in the heart shows that this drug administration carrier is used for the effectiveness increase of target to solid organ.Non-esterified free astaxanthin accumulating in CNS (brain) more not obvious (mean+/-SEM=3.6+ of 4 animals/-1.7nM); Show that it is possible penetrating blood brain barrier (BBB), but long-term, multiple dose administration possibly be that to obtain to use for CNS the protection level of (Alzheimer, apoplexy etc.) necessary.The interaction of the disodium salt disuccinic acid ester derivative of meso astaxanthin and human serum albumin (HSA):

The difference of most of Serlabo carrotenoid and most xenthophylls is water-soluble to have limited their application as water singlet oxygen quencher and free-radical scavengers.The apparent solubility and/or the dispersed chemically modified that will increase carrotenoid are applied to basic science and clinical study.But parent carrotenoid and novel derivative form supramolecular assemblies in the aqueous solution trend makes earlier comprehensive this behavior of thoroughly evaluating just turn to the external and in vivo tests of the effectiveness of these compounds that reasonable ground is arranged then.

Fig. 5 describes carotenoid derivatives, alltrans (complete-E) the synthetic meso astaxanthin of form (3R, 3 ' S-dihydroxyl-β, β-Hu Luobusu-4,4 '-diketone) disodium salt disuccinic acid ester derivative (dAST).Be used to produce the symmetrical C of novel derivative ₄₀-xenthophylls has two chiral centres in 3 and 3 ' position.In the aqueous solution, C ₄₀-xenthophylls does not show optically active, because these three-dimensional centers have opposite absolute configuration, and compensates one another in inside.Has the abundantest protein binding in preferential and human serum albumin (the HSA)-blood of the natural carotenoid molecule of carboxylic acid functional.Because albumin bound is the CBAC in vivo of diving of the given compound of influence strongly, thereby circular dichroism (CD), ultraviolet-visible (UV/Vis) and fluorescence spectrum are used to characterize this novel carotenoid verivate and the not interaction of the HSA of fatty acids.Study the protein binding and the aggregation properties of this symmetry classes Radix Dauci Sativae class, it connects through the part direct esterification that will contain the carboxylic acid end group, forms rigidity, long-chain, highly unsaturated two negatively charged ion bolamphiphile.Proof is not in existing proteic buffered soln, and meso carrotenoid forms the H type card-pack aggregate of the dense packing that does not show CD Cotton effect (CE).But under low part/albumen (L/P) mol ratio; Meso carrotenoid associates with monomer mode and HSA immediately and preferentially; Show that the secondary chemical interaction (Van der Waals force, hydrogen bonding) that supramolecule assembling takes place in permission is overcome in biological relevant environment in the aqueous solution.Surpass 1: 1 part/albumen mol ratio, meso carrotenoid molecule begins to assemble once more; Observed gathering is a chirality under these ratios, produces to show the active supramolecular structure of strong exciton type CD.

The experimental technique summary

By crystallization astaxanthin [3R, 3 ' R, 3R, 3 ' S, 3S, 3 ' S (25: 50: 25)], a kind of statistics mixture of the steric isomer that is purchased (Buckton Scott, India) synthetic new verivate dAST.Separate the astaxanthin steric isomer through HPLC (HPLC), cause the synthetic meso disodium salt disuccinic acid ester derivative that is used for the purifying of this research test.(form is owing in the polyenoid chain of spacer, lacking cis (or Z) configuration thereby being a kind of linearity, stiff molecule (Fig. 5) entirely-E) for the alltrans of used meso steric isomer.Successfully synthesized disodium salt disuccinic acid ester derivative according to the synthetic meso astaxanthin of HPLC purity＞99%.

Material

The human serum albumin (catalog No.A-1887, lot No.14H9319) that is substantially free of lipid acid is available from Sigma, and as use with providing.Use distilled water and spectrum level methyl-sulphoxide (DMSO, Scharlau Chemie S.A., Barcelona, Spain) and ethanol (Chemolab, Budapest, Hungary).All other chemical substances are AG.

The stock solution of preparation dAST

After meso carrotenoid was dissolved in DMSO, being added to 100 μ l DMSO solution at path length was in the 2mL ethanol in the rectangle cuvette of 1cm.Record absorption spectrum under 260-650nm.By at λ _MaxUnder absorbance value calculating concentration (ε _478nm=116,570M ^-1Cm ^-1).

Preparation HSA solution

For the spectra sample preparation, HSA is dissolved in pH 7.4 Ringer or 0.1M pH 7.4 phosphate buffered saline buffers.Calculate albumin concentration, E ^1% _1cm=5.31, use the absorbance data of testing under the 279nm that obtains.The molecular weight of HSA is defined as 66500Da.

Circular dichroism and UV/Vis absorption spectrum

With Jasco J-715 spectropolarimeter, under 25 ± 0.2 and 37 ± 0.2 ℃, be record CD and UV spectrum in the rectangle cuvette of 1cm at path length.Through the Peltier thermostatted that is equipped with magnetic agitation temperature control is provided.With 1.0nm bandwidth and 0.5nm resolving power and scanning speed is the 100nm/ branch, and all spectrum is assembled three times.The CD that inductive CD is defined as the dAST-HSA mixture deducts the identical wavelength CD of only HSA that places an order, and is expressed as the ovality (mdeg) of milli degree.

The dAST that is used under 37 ℃ in pH 7.4 Ringer and the 0.1M phosphate buffered saline buffer carries out the CD/UV/Vis titration to HSA

Will Ringer damping fluid, L/P value are 0.007-0.10: 2mL 1.6 * 10 ^-4It is the cuvette of 1cm that M HSA solution places path length, and adds a small amount of part stock solution (c=2.2 * 10 with 10 μ L aliquots containigs with aupette ^-4).Will The Ringer damping fluid, the L/P value does 0.82-13.13: 2.3 * 10 of 2ml ^-6It is the cuvette of 1cm that M HSA solution places path length, and adds part stock solution (c=3.9 * 10 of μ L volume with aupette ^-4).Will Phosphate buffered saline buffer, L/P value are 0.82-13.10: 2mL 2.2 * 10 ^-6It is the cuvette of 1cm that M HSA solution places path length, and adds part stock solution (c=3.6 * 10 of μ L volume with aupette ^-4).

Measure dAST and have the primary fluorescence of HAS down

In the 1cm rectangular chamber, in 0.1M pH 7.4 phosphate buffered saline buffers, prepare 4.2 * 10 of 2mL ^-6M HSA solution.Continuously with 1.3 * 10 ^-4With 3.3 * 10 ^-4M meso carrotenoid DMSO solution is added in μ L volume in the cuvette in the sample chamber of Jasco J-715 spectropolarimeter.240 and 360nm between excite the sample solution of gained with the 0.5nm wavelength increment.Hamamatsu H5784-type photomultiplier detector with being installed in the light source right corner is collected in the total fluorescence intensity under each wavelength.In sample solution, the initial and ultimate density of HSA and dAST is respectively 4.2 * 10 ^-6M-4.0 * 10 ^-6M and 1.3 * 10 ^-7M-1.4 * 10 ^-5M.Meso carrotenoid/HSA mol ratio changes between 0.03 and 3.53.During fluorometric assay, final DMSO concentration is no more than 5V/V%.Also carry out control experiment, wherein measure the fluorescence that 20,50 and 100 μ L DMSO is added to HSA during the solution.

UV/Vis and the summary of CD spectral results

The UV/Vis of dAST in ethanol and aqueous buffer solution and CD spectral response curve

Because the pi system of its extension, dAST shows strong photoabsorption (Fig. 6) in visible spectrum.It is owing to the minimum energy electronics dipole that allows, along polyenoid chain major axis polar π → π * transition that main bell absorption band concentrates on the 481.5nm place.Under the room temperature, for the carrotenoid that comprises one or more conjugation carbonyls, it is typical lacking fine structure.But the vibration sub-band actually exists under this curve, as by the secondary announcement (Fig. 6) of deriving of spectrographic.In addition, there is further transition in nearly UV district.According to the Theoretical Calculation of on the polyenoid model, carrying out, near the transition of electron square (μ) of the bands of a spectrum of the suitable intensity 300nm is parallel to the major axis polarization of dAST molecule.Simultaneously, the bands of a spectrum μ at 371nm place orientation is along the dual C of conjugated system ₂Symmetry axis.The weak n of carbonyl → π * transition is owing to other bands of a spectrum thicken.As the expection, the meso carotinoid compounds does not show any CD bands of a spectrum in ethanol because two opposite chiral centres ((data not shown) cancelled in the effect of 3R 3 ' S) each other.

In the Ringer damping fluid, the main absorption band of dAST changes, and shows big blue displacement (2541.6cm ^-1) and frequency range constriction (Fig. 7).These spectrum change show and form so-called " card-pack " aggregate, and molecule wherein is through be closely linked (in the several dust scopes) that interact with the H bonding of the exclusion from aqueous environments.The result is that the excite state wave energy of polyenoid chain allows between adjacent molecule, to take place the exciton Resonant Interaction in intermolecular generation delocalization.This interaction causes the high-energy exciton peak in the UV/Vis spectrum.Owing to disadvantageous steric interaction in huge end group, occurs, do not allow being arranged in parallel of polyenoid chain; The major axis of molecule replaces and seals the intermolecular angle of coverage of confirming separately.In these cases, the carotenoid aggregates that is made up by chiral monomer also shows inductive Cotton effect (CE) owing to arranging between the chiral molecules that is determined by asymmetric center.By contrast, the meso carotinoid compounds does not show optically active (data not shown) in solution under state of aggregation, and reason is to lack clean molecule handedness.

The optical characteristics of dAST in the presence of the human serum albumin of low part/albumen mol ratio

When in the HSA solution that in pH 7.4 Ringer damping fluids, prepares, adding dAST, two inductive CD bands of a spectrum clear and definite, contrary sign appear at 300 and 450nm between, and zero cross point is at 367nm (Fig. 8).Accompanying drawing inserts intensity and the main absorption band under Different L/P ratio (Δ ε and ε value are calculated with respect to total meso carotenoid concentration) that shows the inductive Cotton effect.The size of CEs increases with ligand concentration, but their shape and wavelength location remain unchanged.As above-mentioned, below 450nm, there are two transition, it possibly be responsible for observed optically active.Near the 300nm absorption band has transition symmetry B, and corresponding electricity and magnetic transition square are vertical with dual symmetry axis along the polyenoid chain.Electricity and magnetic transition square and C with the bands of a spectrum at 372.5nm place ₂The axle parallel polarization, its transition is to being called A.Can infer reasonably that when protein binding these bands of a spectrum are owing to the change of microenvironment around the polyenoid chain is moved to longer wavelength.Prove that well (chromophoric group partly belongs to C for the CD spectrum of carrotenoid ₂The point group) meets C ₂-rule: if total conjugation system obtains right hand chirality (being that key 6-7 and 6 '-7 ' dihedral angle is on every side born), then the transition of symmetrical A causes negative CE, and the transition of symmetrical B causes positive CE (Fig. 8).Therefore, meso carrotenoid combines with HSA by this way: the albumen environment end-rings of definite chiral conformation that is fixed well, thus cause observing negative, positive inductive CD bands of a spectrum.

The absolute configuration at chirality 3 and 3 ' center does not determine the chirality optical characteristics of molecule; On the contrary, the asymmetric albumen environment (through non-covalent chemical interaction) of BSA molecule determines observed activity.Compare with above-mentioned gathering behavior in the aqueous solution, the dAST molecule is not assembled in the HSA of these L/P ratios solution, and this maintenance through the visible absorbance bands of a spectrum of bell and slight red displacement proves (Fig. 8).Like this UV/Vis absorb and CD spectrum all show meso carrotenoid molecule and HSA combine take place with monomeric form.

Surpass 1: there is the optical characteristics of dAST down in the HSA of 1L/P ratio

The HSA solution that the dAST of increasing amount is added to pH 7.4 Ringer or the preparation of 0.1M pH 7.4 phosphate buffered saline buffers is higher than 1 to realize the L/P ratio.CD and UV/Vis absorption spectrum all show noticeable change (Fig. 9 and Figure 10) during adding part.Except the visible absorbance bands of a spectrum of blue displacement, one new just-negative CD bands of a spectrum to appear at respectively 480 and 420nm near.These CE ' s do not show the fine structure of vibration, and their amplitude increases with the increase of ligand concentration.But, there is some marked difference between the spectrum that in Ringer and phosphate buffered saline buffer, obtains:

A) main absorption band migrates to lower wavelength (434.5nm) in the Ringer damping fluid.Analog value in the phosphate buffered saline buffer is 451.5nm.

B) Ringer (441.6cm ^-1) in from the deviation ratio phosphate buffered saline buffer (148.4cm of the zero cross point of the CEs of maximum absorption bands of a spectrum ^-1) big 3 times.

C) the L/P value is more than 8, and the intensity of the CD bands of a spectrum in the Ringer solution no longer increases.By contrast, the amplitude of CD bands of a spectrum continues to increase with L/P ratio in the phosphate buffered saline buffer, even when the L/P value is 13.

D) under identical L/P ratio, in phosphate buffered saline buffer, measure more intensive CD bands of a spectrum (Fig. 9 and Figure 10).

The CD bands of a spectrum of these contrary signs are only surpassing 1: the fact that occurs during the 1L/P ratio shows that consumingly they are derived from the chiral molecules interphase interaction between the adjacent meso carrotenoid molecule.When two transition of electron moment of dipole energy similar; Spatially close to each other; And when forming chirality and arranging; Their interaction shows as the coupling of chirality exciton: CD spectrum shows bisignate couplet, is complementary with the spectral position of corresponding absorption band, and the signal of said absorption band is confirmed by the absolute sense of the distortion between two dipoles.According to exciton chirality rule, just twisting negative CE corresponding to positive long wavelength CE and shorter wavelength place, vice versa.Under our situation, the direction of transition dipole moment is known; It is along the major axis polarization of polyenoid chain.Therefore, adjacent meso carrotenoid molecule is so that their major axis is just forming the mode of (clockwise) intermolecular angle of coverage arranges.The chirality of two conjugated chains shown in Figure 11 is arranged and is satisfied the former condition; In these cases, the long wavelength just will appear in the CD spectrum with the negative bands of a spectrum of short wavelength.But the spectroscopy performance of absorption band helps to distinguish these spatial disposition.Owing between the transition dipole moment of adjacent meso carrotenoid molecule under the situation of a and b, have disadvantageous Coulomb interactions (Figure 11), absorb peak and be moved to higher energy; If the c form exists, then absorption band broadens and its peak is moved to lower energy.Therefore, the dAST molecule forms right hand chirality and arranges, and wherein the monomeric major axis of meso carrotenoid forms positive acute angle (Figure 11, a and b).

The following imagination of the chirality ordering origin of relevant ligand molecular is proposed.BSA seemingly induces optical activity necessary, and at first interesting is to infer on HSA, to have the big binding site that can hold two meso carrotenoid molecules.BSA will only combine single part under low L/P value; Under higher L/P concentration, with the compound second meso carrotenoid monomer.But as above-mentioned, the size of CEs continues to increase (Figure 10) under quite high L/P value, and single in this case binding site should be by saturated.A solution of this problem infers that HSA is a kind of asymmetric template, begins the chirality self-assembly above that.Some meso carrotenoid molecules the earliest combine with HSA with right hand property arrangement mode, and meso carrotenoid monomer subsequently is based upon on this chiral structure.In this imagination, HSA provides first basic step, and chirality causes (" chirality inoculation "); After this self-assembly automatically continues.But extremely important noticing of being, under the situation that does not have its chirality end group, only minority dAST molecule keeps the arrangement of right hand property at the binding site place of HSA.3 and 3 ' chiral centre play a decisive role aspect the chirality self-chambering part allowing aggregate on the HSA molecule, to form.Do not exist under the proteic situation, meso carrotenoid molecule is owing to the degree that the difference of shortage chirality forms the right hand and left-handed assembly equates.

As above-mentioned, the SPECTRAL DIVERSITY between the CD curve of in phosphate buffered saline buffer and Ringer solution, measuring shows the influence (Fig. 9 and Figure 10) of salt concn to aggregate stability.The osmolarity and the ionic strength of Ringer damping fluid are higher than phosphate buffered saline buffer.Ionize all takes place 7.4 times at pH in the succsinic acid part in two kinds of damping fluids, between aggregate inside and aggregate, all form Coulomb repulsion.The salt ion of positively charged can reduce this repulsion, thereby helps under these cationic situation of existence, to increase the stable and big or small of aggregate.Using above 1: during the dAST titration HSA of 1L/P ratio, form chirality and achirality aggregate simultaneously; But only chirality aggregate and HSA associate, and the achirality aggregate does not then have.It is better stable that the CD spectrum (Fig. 9) that in Ringer buffered soln, obtains shows that the achirality aggregate is able in this higher osmolarity damping fluid owing to the masking effect of salt ion.The ligand molecular that adds is preferential to associate with the aggregate that exists, thereby causes the amplitude of CD bands of a spectrum to reach platform, and becomes constant, forms contrast with phosphate buffered saline buffer.

The quenching of fluorescence of HSA behind the adding dAST

The single tryptophan residue (Trp214) that is positioned at subdomain IIA depths is responsible for the primary fluorescence of HSA to a great extent.The absorption spectrum of the fluorescence emission spectrum of HSA and meso carrotenoid is overlapping.Therefore, after the solution that will the dAST increment in DMSO joins HSA, obtaining fluorescence spectroscopy measures.The result clearly illustrates that meso-carrotenoid molecule primary fluorescence of quencher HSA (Figure 12) effectively.The DMSO that is used to prepare the stock solution of dAST can ignore (Figure 12) to intrinsic HAS fluorescence influence.Under 0.7 L/P ratio, the baseline fluorescence intensity reduces 50%.Viewed phenomenon show meso carrotenoid molecule be combined in Trp214 near, form a part (site I, the subdomain IIA of wall among its in two main binding cavities of HSA; Figure 13).But, site I and site II (subdomain IIIA)-all be hydrophobic fatty acids combine passage-all can not hold long, rigidity dAST molecule (Figure 13).Based on structural similarity, second kind of possibility is that dAST combines with other long-chain (C18, C20) lipid acid binding site of HSA, and said site characterizes with high resolving power X-radiocrystallgraphy fully.Under the situation of the short open chain carrotenoid that does not have huge end group, this possibility is possible.But the polyenoid chain survey of meso carotenoid derivatives itself is

(3 and 3 ' chiral carbon atom between).Although their conformation mobility; Succsinic acid partly needs additional space; The useful length of molecule is increased to

and check that carefully the crystalline structure of HSA shows, the length between structural domain I and the III, narrow crack possibly be suitable for combining meso carrotenoid molecule (Figure 13).The crack is wide between the territory, and its narrow end is near tryptophane (Trp214; * on Figure 13) residue, this observed quenching of fluorescence when the crack combines between the territory of meso carrotenoid molecule and HSA provides interpretation of structure.And, can infer that the significant conformational change of HSA is induced in the association of the single molecule between additional dAST molecule and territory in the crack, causes central slit to broaden.This possibly be that quenching of fluorescence does not stop at the L/P=1 ratio, and increases the reason (Figure 13) that continues reinforcement with CEs.

UV/Vis and CD spectroscopy result are discussed

As the result who is ostracised from aqueous environments with the intermolecular ydrogen bonding bonding; The disodium salt disuccinic acid ester derivative of synthetic achirality meso astaxanthin forms the aggregate of the photoactive card-pack type of irrotationality in aqueous buffer solution, this BigBlue displacement through main visible absorbance bands of a spectrum with respect to observed bands of a spectrum in ethanolic soln shows.As if under the situation of the HSA that has excessive not fatty acids, meso carrotenoid preferentially associates with monomer mode and HSA.These results show that the weak Van der Waals force and the hydrogen bonding that allow the supramolecule assembling takes place will be overcome rapidly in biological relevant environment in the aqueous solution.Albumin concentration in the human blood is about 0.6mM in vivo; Be illustrated under the dosage of as many as 500mg; Meso carrotenoid (molecular weight 841Da) will be with monomer mode and BSA association (the extra potential non-specific binding of eliminating and circulation hemocyte and lipoprotein, this can increase potential non-gathering dosage).Bonded meso carrotenoid molecule shows inductive CD bands of a spectrum, and this bands of a spectrum are fully explained with the right hand property helical conformation of conjugated system.Classification quenching of fluorescence increasing HSA in the presence of the dAST of concentration has been strengthened such viewpoint: this quencher is responsible in the formation of carrotenoid-BSA complex body, and shows the spatial neighbor between tryptophane 214 residues of bonded part and HSA.

Based on the molecular length of spectroscopic data, dAST molecule and the crystalline structure of the abundant HSA that characterizes, binding site is specified in crack between the territory between structural domain I and the III tentatively.

Be higher than 1: as if just exist in the meso carrotenoid of 1L/P ratio and the CD spectrum of HSA-negative bands of a spectrum are right.This discovery is owing to the intermolecular chirality exciton coupling between the meso carrotenoid polyenoid chain of arranging with the assembling of right hand property.Experimental data shows that HSA is used as a kind of chiral template, and self-assembly begins on this template, continues then, arranged by the chirality of the end group of meso carrotenoid molecule.Difference between the bisignate CD spectrum that in pH 7.4 phosphate buffered saline buffers and Ringer solution, obtains shows that self-assembly receives the osmolarity and the ionic strength affect of solution.Along with osmolarity increases, the stability of aggregate improves, and presumably is owing to the electrostatic shielding of salt cation to electronegative succsinic acid carboxylic acid functional.

The further modification of different aspect of the present invention and the embodiment of alternative selection consider that this specification sheets is apparent to those skilled in the art.Therefore, should this specification sheets be interpreted as it only is exemplary, its objective is the general fashion of instruction those skilled in the art embodiment of the present invention.Should be appreciated that, should be with being interpreted as present embodiment preferred with described form of the present invention shown in this paper.Can substitute the element and the material of this paper illustration and description, part and process can be put upside down, and some characteristic of the present invention can utilize independently, and all these are conspicuous to the those skilled in the art that from description of the invention, benefited.Can under the situation that does not deviate from described essence of the present invention of following claim and scope, change key element as herein described.

Claims (7)

1. the compound that has following structure:

Wherein

Each Y is O or H independently ₂

Each R is R ¹

Each R ¹Be independently-alkyl-NR ² ₃ ⁺, or-amino acid-NH ₃ ⁺And

Each R ²Be H, alkyl or aryl independently.

2. the compound of claim 1, wherein R is-amino acid-NH ₃ ⁺

3. the compound of claim 1, wherein R is-alkyl-NR ² ₃ ⁺

4. pharmaceutical composition, it comprises compound and pharmaceutically acceptable carrier or the vehicle of claim 1～3 described in each.

5. the compound of claim 1～3 described in each is used for treating the purposes of the medicine of ischemia-reperfusion injury in preparation.

6. the compound of claim 1～3 described in each is used for treating the purposes of the medicine of cancer cells and precancerous cell in preparation.

7. the compound of claim 1～3 described in each is used for treating the purposes of the medicine of hepatic diseases in preparation.

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